Photoactivatable Antimicrobial Agents And Therapeutic And Diagnostic Methods Of Using Same

ABSTRACT

The present invention provides photosensitizer compounds for use in detecting beta-lactamase activity. Methods and kits that utilize the photosensitizer compounds of the invention for the detection of, quantitation of, and classification or typing of microbial beta-lactamases.

CROSS-REFERENCE TO RELATED APPLICATIONS/PATENTS & INCORPORATION BYREFERENCE

This application claims priority to U.S. Provisional application Ser.No. 61/050,453, filed May 5, 2008.

This application is also related to International application No.PCT/US2006/044369, filed Nov. 15, 2006, and to U.S. Provisionalapplication Ser. No. 60/736,917, filed Nov. 15, 2005. All of theaforementioned Provisional and International patent applications arehereby expressly incorporated herein by reference in their entireties.

Any and all references cited in the text of this patent application,including any U.S. or foreign patents or published patent applications,International patent applications, as well as, any non-patent literaturereferences, including any manufacturer's instructions, are herebyexpressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

The widespread use of antimicrobial chemotherapeutics has had theinevitable consequence of the emergence of antibiotic-resistantpathogens, which has continualy prompted further development and designof new drugs to combat such organisms. Today, more than 70% of thebacteria associated with nosocomial infections in the United States areresistant to one or more of the drugs previously used to treat them.Drug resistance in bacteria—which is not limited to the U.S. but extendsthroughout the world—includes, for example, the worldwide emergence ofHaemophilus and gonococci that are resistant to β-lactam antibiotics(e.g., penicillin), methicillin-resistant Staphylococcus aureus,multiple-drug resistant S. aureus with high-level resistance tovancomycin, various isolated strains of Pseudomonas and Enterobacterthat are resistant to all available antibiotics, and multiple-drugresistant strains of Mycobacterium tuberculosis.

While society increasingly has recognized the negative consequences ofthe misuse of antibiotics, overuse and overprescribing of antibioticscontinue to be widespread throughout the world, driven by diagnosticuncertainties, demands by patients, and physicians' lack of time toeffectively evaluate patients. Although reducing inappropriateantibiotic use is thought to be the best way to curb resistance,physicians must generally be more selective and prudent in their use andprescribing of antibiotics so that the gains in the battle againstinfectious diseases over the past century are not lost. The rampantspread of antibiotic resistances mandates a more responsible andsensible approach to antibiotic use.

The β-lactam antibiotics (e.g., β-lactam ring-containing antibiotics,such as, penicillins, cephalosporins, or carbapenems, which inhibitbacterial cell wall synthesis) are a particularly prevalent andimportant class of antibiotics that are widely prescribed for a largevariety of gram-negative and gram-positive infections. Consequently,widespread resistance has emerged. One particularly important mechanismof microbial resistance to the β-lactam antibiotics stems generally fromthe production of enzymes known as the β-lactamases orcephalosporinases, which enzymatically cleave β-lactam antibioticsthereby causing their inactivation. This type of resistance can beencoded on the chromosome or on extra-chromosomal elements (e.g.bacterial plasmids), which can be transferred horizontally to otherbacteria. Other modes of resistance to β-lactam antibiotics also includeacquisition of penicillin-binding proteins and decreased entry and/oractive efflux of drugs through membrane efflux pump systems.

Clinical detection of β-lactamases represents a key step in themanagement of antibiotic therapy of bacterial infections. In particular,the amount of beta-lactamase activity and the substrate specificity ofthat activity are important considerations in determining theappropriate antibiotic therapy for patients suffering from drugresistant bacterial infections.

β-lactam susceptibility and resistance can be detected and/or measuredin a variety of ways. For example, Kirby-Bauer antibiotic testing usesantibiotic-impregnated discs to test whether particular bacteria aresusceptible to specific antibiotics. A known quantity of bacteria isgrown on agar plates in the presence of thin wafers containing relevantantibiotics, such as penicillin or ampicillin. If the bacteria aresusceptible to the antibiotics, a zone of inhibition forms around thediffusion zone of the wafers. The size of the zone is correlated to theminimum inhibitory concentratin (“MIC”) of antibiotic for that bacteria.In this way, health care providers are able to choose appropriateantibiotics to combat a particular infection.

In addition, agar dilution methods and broth microdilution methods canbe used. Many of these labor-intensive and time-consuming methods oftenfail to detect drug resistance in certain gram-negative bacteria and allrequire a pregrowth step in which the strain is grown in broth or on aplate under conditions in which the organism is exposed only to theinducing antibiotic. This step is followed by a challenge in thepresence of an indicator antibiotic or direct assay of enzymaticactivity. These approaches require pure culture inoculation and growth,and involve up to 24 hours of incubation.

Chromogenic substrates have also been used, which, when cleaved bybacterial beta-lactamases, produce a colorimetric change that can bedetected or measured. Examples of such substrates include, for example,nitrocefin and centa, which are known in the art. Nitrocefin is sold inthe form of impregnated paper discs, which, when placed in the vicinityof a bacterial culture producing β-lactamase, results in the developmentof a pink color. Although this method provides a rapid qualitativedetection (i.e., yes/no), it does not provide any information regardingthe relative amount of enzymatic activity or any insight into the typeof beta-lactamase activity, and, thus, cannot be used alone to determinethe appropriate course of therapy in a clinical setting.

As can be seen, currently available methods for detecting and evaluatingbeta-lactamase activity and resistance to beta-lactam antibiotics suffera number of drawbacks. In particular, such methods, while providingqualitative information (yes/no) about drug resistance, aretime-consuming and do not easily facilitate the quantitative measurementof enzyme activity or determination of enzyme type or substratecharacteristics—which constitute highly valuable information fordetermining an appropriate strategy for antimicrobial therapy.

Accordingly, new methods and compositions for detecting and evaluatingbeta-lactamase activity, which are more sensitive, rapid and easier toperform, and which reliably and expediently provide both qualitative andquantitative information as to the activity and/or substrate specificityof a beta-lactamase, would represent an advance in the art.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions that can beadvantageously utilized for the qualitative and/or quantitativedetection of beta-lactamase activity, which are more sensitive, rapidand easier to perform than prior methods. The methods and compositionsof the invention can also be used to evaluate the substrate specificityof a beta-lactamase activity from any sample containing a beta-lactamaseenzyme, including, for example, bodily fluids, cells or tissues carryingan infection with a pathogenic beta-lactamase-producing bacterium. Theinvention advantageously provides a method to determine effective andsuitable strategies for the treatment of bacterial infections usingappropriate regimes of antibiotics, which are effective against targetorganisms.

In one aspect, the present invention provides a photosensitizercomposition, wherein the composition comprises at least onebenzophenothiazinium chloride (EtNBS) photosensitizer conjugated to acephalosporin linker or fragment thereof. The composition can includetwo benzophenothiazinium chloride (EtNBS) photosensitizers conjugated toa cephalosporin linker (L) or fragment thereof. The photosensitizer canbe bound at the 3′ position of a cephalosporin. In one aspect, thephotosensitizers are quenched when the linker is uncleaved.

In another aspect, the present invention provides a photosensitizercomposition according to formula I:

X-L-X′,

wherein L is a cephalosporin linker or fragment thereof, X isbenzophenothiazinium chloride (EtNBS) and X′ is benzophenothiaziniumchloride (EtNBS), wherein the photosensitizers are in a quenched statewhen L is uncleaved.

The photosensitizer composition can further include a targeting moiety,which can target the composition to a pathogen or a host cell, e.g., amacrophage, infected with a pathogen. The targeting moiety can include aliposome, a peptide, or a small anti-microbial peptide or an activefragment or analog thereof.

The photosensitizer composition of the invention can also include one ormore binders effective to quench photoactivation of thebenzophenothiazinium chloride (EtNBS). The binder can be a fluorophoreor a photosensitizer in various aspects.

In still another aspect, the photosensitizer composition of theinvention can further include a backbone coupled to the twobenzophenothiazinium chloride (EtNBS) and one or more binders effectiveto quench photoactivation, wherein the binders are connected to thebackbone through the linker. The backbone can include a targeting moietyand can be a polyamino acid (e.g., polylysine) in one aspect.

The L (or linker) can include a penicillin, such as, benzthinepenicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin(penicillin V), procaine penicillin, oxacillin, methicillin,dicloxacillin, flucloxacillin, temocillin, amoxicillin, ampicillin,co-amoxiclav, carboxypenicillins, ureidopenicillins, azlocillin,carbenicillin, ticarcillin, mezlocillin, piperacillin, or any fragmentor derivative of the above. The L can also include a cephalosporink,such as, (cephacetrile), Cefadroxil (cefadroxyl; Duricef®), Cefalexin(cephalexin; Keflex®), Cephaloglycin, Cefalonium (cephalonium),Cefaloridine (cephaloradine), Cefalotin (cephalothin; Keflin®),Cefapirin (cephapirin; Cefadryl®), Cefatrizine, Cefazaflur, Cefazedone,Cefazolin (cephazolin; Ancef®, Kefzol®), Cefradine (cephradine;Velosef®), Cefroxadine, Ceftezole, Cefaclor (e.g., Ceclor®, Distaclor®,Keflor®, Raniclor®), Cefonicid (e.g, Monocid®), Cefprozil (e.g.,cefproxil; Cefzil®), Cefuroxime (e.g., Zinnat®, Zinacef®, Ceftin®,Biofuroksym®), Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin,Carbacephems (e.g., loracarbef (Lorabid®)), Cephamycins (e.g.,cefbuperazone, cefmetazole (Zefazone®), cefrninox, cefotetan (Cefotan®),cefoxitin (Mefoxin®)), cefotetan or cefoxitin, Cefcapene, Cefdaloxime,Cefdinir (Omnicef®), Cefditoren, Cefetamet, Cefixime (Suprax®),Cefmenoxime, Cefodizime, Cefotaxime (Claforan®), Cefpimizole,Cefpodoxime (Vantin®, PECEF), Cefteram, Ceftibuten (Cedax), Ceftiofur,Ceftiolene, Ceftizoxime (Cefizax®), Ceftriaxone (Rocephin®),Cefoperazone (Cefobid), Ceftazidime (Forturn®, Fortaz®), or Oxacephems(e.g. latamoxef), cefepime (Maxipime®), cefclidine, cefluprenam,cefoselis, cefozopran, cefpirome, cefquinome, cefpirome, or a fragmentof derivative of any of the above composition further comprises atargeting moiety.

In another aspect, the present invention provides a method for detectinga beta-lactamase activity in a sample, comprising the steps of:contacting the sample with a photosensitizer composition comprising twoor more photosensitizers that are conjugated to a linker, wherein thelinker comprises a cleavage site for a beta-lactamase and wherein thephotosensitizers are quenched when the cleavage site is intact butunquenched when the cleavage site is hydrolyzed; and detecting cleavageof said linker, wherein cleavage of said linker is indicative of abeta-lactamase activity in the sample.

In still a further aspect, the present invention provides a method fordetermining the substrate specificity of a beta-lactamase enzyme in asample, comprising: contacting the sample with a photosensitizercomposition comprising two photosensitizers that are conjugated to alinker, wherein the linker comprises a cleavage site for abeta-lactamase enzyme and wherein the photosensitizers are quenched whenthe cleavage site is intact but unquenched when the cleavage site iscleaved; and determining whether the linker is cleaved, wherein cleavageof the linker indicates the linker is a substrate of the beta-lactamaseenzyme.

The invention also provides, in another aspect, a method of typing abeta-lactamase enzyme in a sample, comprising: performing a competitivereaction comprising the steps of (a) contacting the sample with aphotosensitizer composition comprising two photosensitizers that areconjugated to a linker, wherein the linker comprise a cleavage site fora beta-lactamase and wherein the photosensitizers are in a quenchedstate; (b) cleaving the linker to dequench the photosensitizers; (c)light-activating the composition to produce a fluorescence signal; and(d) quantifying the fluorescence signal with a detector to obtain aresult, said competitive reaction being performed in the presence of acompeting beta-lactam antibiotic; and comparing the result of thecompetitive reaction to a standard to type the beta-lactamase. Thestandard can be determined by performing a non-competitive reactioncomprising the steps of (a) contacting the sample with a photosensitizercomposition comprising two photosensitizers that are conjugated to alinker, wherein the linker comprise a cleavage site for a beta-lactamaseand wherein the photosensitizers are in a quenched state; (b) cleavingthe linker to dequench the photosensitizers; (c) light-activating thecomposition to produce a fluorescence signal; and (d) quantifying thefluorescence signal with a detector to obtain a standard.

In certain aspects, the step of detecting an unquenched photosensitizercan include detecting a signal produced by the unquenchedphotosensitizer. The signal can be a fluorescence emission induced byilluminating the unquenched photosensitizer with an excitationwavelength.

In other aspects, the sample can be a biological sample isolated from aninfection in a subject, such as a mammal, including a human. Theinfection can be caused by an antibiotic-resistant pathogen, which canbe a Gram (−) bacterium or Gram (+) bacterial pathogen.

In some aspects, the antibiotic-resistant pathogen can be aStaphylococcus, Enterococcus, Enterobacter, Escherichia, Haemophilus,Neisseria, Klebsiella, Pasteurella, Proteus, Pseudomonas,Streptophomonas, Burkholderia, Acinetobacter, Serratia, or Salmonellaspp.

The antibiotic-resistant pathogen can also be a Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, or Salmonellatyphimurium.

In certain aspect, the photosensitizer can be a porphryin, including aporfimer sodium, hematoporphyrin IX, hematoporphyrin ester,dihematoporphyrin ester, synthetic diporphyrin, O-substitutedtetraphenyl porphyrin, 3,1-meso tetrakis porphyrin, hydroporphyrin,benzoporphyrin derivative, benzoporphyrin monoacid derivative, monoacidring derivative, tetracyanoethylene adduct of benzoporphyrin, dimethylacetylenedicarboxylate adduct of benzoporphyrin, δ-aminolevulinic acid,benzonaphthoporphyrazine, naturally occurring porphyrin, ALA-inducedprotoporphyrin IX, synthetic dichlorin, bacteriochlorintetra(hydroxyphenyl) porphyrin, purpurin, octaethylpurpurin derivative,etiopurpurin, tin-etio-purpurin, porphycene, chlorin, chlorin e₆,mono-1-aspartyl derivative of chlorin e₆, di-1-aspartyl derivative ofchlorin e₆, tin(IV) chlorin e₆, meta-tetrahydroxyphenylchlorin, chlorine₆ monoethylendiamine monamide, verdin, zinc methyl pyroverdin, copro IIverdin trimethyl ester, deuteroverdin methyl ester, pheophorbidederivative, pyropheophorbide, texaphyrin, lutetium (III) texaphyrin, orgadolinium(III) texaphyrin.

other aspects, the photosensitizer can be a photoactive dye, including amerocyanine, phthalocyanine, chloroaluminum phthalocyanine, sulfonatedaluminum PC, ring-substituted cationic PC, sulfonated AlPc, disulfonatedor tetrasulfonated derivative, sulfonated aluminum naphthalocyanine,naphthalocyanine, tetracyanoethylene adduct, crystal violet, azure βchloride, benzophenothiazinium, benzophenothiazinium chloride (EtNBS),phenothiazine derivative, rose Bengal, toluidine blue derviatives,toluidine blue O (TBO), methylene blue (MB), new methylene blue N(NMMB), new methylene blue BB, new methylene blue FR,1,9-dimethylmethylene blue chloride (DMMB), methylene blue derivatives,methylene green, methylene violet Bernthsen, methylene violet 3RAX, Nileblue, Nile blue derivatives, malachite green, Azure blue A, Azure blueB, Azure blue C, safranine O, neutral red,5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,thiopyronine, or thionine.

In one particular aspect, the photosensitizer composition includes twobenzophenothiazinium chloride (EtNBS) photosensitizers conjugated to acephalosporin linker or fragment thereof.

In certain other aspects, the methods of the invention include the stepof quantitating the signal produced by the unquenched photosensitizer.The step of quantitating the signal can include measuring the amount ofthe signal with a detector.

In yet another aspect, the invention provides a kit for detecting abeta-lactamase activity in a sample comprising a photosensitizercomposition of the invention and instructions for using thephotosensitizer composition to detect a beta-lactamase activity in asample.

The invention also provides, in another aspect, a kit for determiningthe substrate specificity of a beta-lactamase activity in a samplecomprising the photosensitizer composition of the invention andinstructions for using the photosensitizer composition to type abeta-lactamase activity in a sample.

In still another aspect, the invention provides a method of treating abacterial infection in a subject in need, comprising administering atherapeutically effective amount of an antibiotic, wherein theantibiotic does not have the same structure as a cleavable linkerdetected by any of the methods of the invention.

The present invention provides, in one aspect, a photosensitizercomposition comprising a plurality of photosensitizers that areconjugated to a linker, wherein the linker includes a cleavage site fora beta-lactamase and wherein the photosensitizers are in a quenchedstate. In one aspect, the linker includes a cephalosporin, a penicillin,a penem, a carbapenem, a monocyclic mobactem, a polypeptide cleavable byan enzyme of Leishmania, or a fragment thereof. In another aspect thelinker comprises a beta-lactam ring or functional derivative thereof. Inyet another aspect, the linker is conjugated to two photosensitizers.

According to several aspects of the invention, the cleavage site of thelinker is a substrate of a beta-lactamase of a pathogen. The pathogencan be a Gram (−) bacterium or Gram (+) bacterium. In certain aspects,the pathogen can be Staphylococcus, Enterococcus, Enterobacter,Escherichia, Haemophilus, Neisseria, Klebsiella, Pasteurella, Proteus,Pseudomonas, Streptophomonas, Burkholderia, Acinetobacter, Serratia, orSalmonella spp. The pathogen can particularly be Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, and Salmonellatyphimurium.

The photosensitizers of the photosensitizer compositions of theinvention, in certain aspects, can be a porphyrin, which can includeporfimer sodium, hematoporphyrin IX, hematoporphyrin ester,dihematoporphyrin ester, synthetic diporphyrin, O-substitutedtetraphenyl porphyrin, 3,1-meso tetrakis porphyrin, hydroporphyrin,benzoporphyrin derivative, benzoporphyrin monoacid derivative, monoacidring derivative, tetracyanoethylene adduct of benzoporphyrin, dimethylacetylenedicarboxylate adduct of benzoporphyrin, δ-aminolevulinic acid,benzonaphthoporphyrazine, naturally occurring porphyrin, ALA-inducedprotoporphyrin IX, synthetic dichlorin, bacteriochlorintetra(hydroxyphenyl) porphyrin, purpurin, octaethylpurpurin derivative,etiopurpurin, tin-etio-purpurin, porphycene, chlorin, chlorin e₆,mono-1-aspartyl derivative of chlorin e₆, di-1-aspartyl derivative ofchlorin e₆, tin(IV) chlorin e₆, meta-tetrahydroxyphenylchlorin, chlorine₆ monoethylendiamine monamide, verdin, zinc methyl pyroverdin, copro IIverdin trimethyl ester, deuteroverdin methyl ester, pheophorbidederivative, pyropheophorbide, texaphyrin, lutetium (III) texaphyrin, orgadolinium(III) texaphyrin.

In other aspects, the photosensitizer can be a photoactive dye, such as,a merocyanine, phthalocyanine, chloroaluminum phthalocyanine, sulfonatedaluminum PC, ring-substituted cationic PC, sulfonated AlPc, disulfonatedor tetrasulfonated derivative, sulfonated aluminum naphthalocyanine,naphthalocyanine, tetracyanoethylene adduct, crystal violet, azure βchloride, benzophenothiazinium, benzophenothiazinium chloride (EtNBS),phenothiazine derivative, rose Bengal, toluidine blue derviatives,toluidine blue O (TBO), methylene blue (MB), new methylene blue N(NMMB), new methylene blue BB, new methylene blue FR,1,9-dimethylmethylene blue chloride (DMMB), methylene blue derivatives,methylene green, methylene violet Bernthsen, methylene violet 3RAX, Nileblue, Nile blue derivatives, malachite green, Azure blue A, Azure blueB, Azure blue C, safranine O, neutral red,5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,thiopyronine, or thionine.

In still further aspects, the photosensitizer can be a Diels-Alderadduct, dimethyl acetylene dicarboxylate adduct, anthracenedione,anthrapyrazole, aminoanthraquinone, phenoxazine dye, chalcogenapyryliumdye, cationic selena, tellurapyrylium derivative, cationic imminium saltor tetracycline.

In a particular aspect, the photosensitizer compositions of theinvention can include two benzophenothiazinium chloride (EtNBS)photosensitizers conjugated to a cephalosporin linker or fragmentthereof.

In certain other aspects, the photosensitizer compositions hereindescribed can include a targeting moiety, which can target a compositionto a pathogen or a host cell infected with a pathogen, or to a liposomeor to a protein (e.g., cell surface protein).

In additional aspects, the photosensitizer compositions herein describedcan include one or more binders, which are effective to quenchphotoactivation of the one or more photosensitizers of the invention. Abinder, which can also be called a quencher, can be a fluorophore,another photosensitizer or like compound.

In certain other aspects, the photosensitizer compositions of theinvention can include a backbone which is coupled to the one or morephotosensitizers, one or more binders (if present), or one or moretargeting moieties (if present). The backbone can be, for example, apolyamino acid or polylysine.

In another aspect, the present invention provides a photosensitizercomposition according to formula I:

X-L-X′,

wherein L is a linker comprising a beta-lactamase cleavage site, X is afirst photosensitizer and X′ is a second photosensitizer, wherein thephotosensitizers are in a quenched state.

In one aspect, the linker can be a cephalosporin, a penicillin, a penem,a carbapenem, a monocyclic mobactem, a polypeptide cleavable by anenzyme of Leishmania, or a fragment thereof, or a compound containing abeta-lactam ring.

The cleavage site of the linker can be a substrate of a beta-lactamaseof a pathogen. The pathogen can be a Gram (−) bacterium or Gram (+)bacterium. The pathogen can also be Staphylococcus, Enterococcus,Enterobacter, Escherichia, Haemophilus, Neisseria, Klebsiella,Pasteurella, Proteus, Pseudomonas, Streptophomonas, Burkholderia,Acinetobacter, Serratia, or Salmonella spp, or more particularly, can beStaphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Haemophilus influenzae,Neisseria gonorrhea, Klebsiella pneumoniae, Pasteurella multocida,Proteus mirabilis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia,Burkholderia cepacia, Acinetobacter baumannii, Enterobacter aerogines,Enterobacter cloacae, Serratia marcescens, Salmonella enterica, orSalmonella typhimurium.

In another aspect, one or both of the photosensitizers, X and X′, can bea porphyrin. A porphyrin can be a porfimer sodium, hematoporphyrin IX,hematoporphyrin ester, dihematoporphyrin ester, synthetic diporphyrin,O-substituted tetraphenyl porphyrin, 3,1-meso tetrakis porphyrin,hydroporphyrin, benzoporphyrin derivative, benzoporphyrin monoacidderivative, monoacid ring derivative, tetracyanoethylene adduct ofbenzoporphyrin, dimethyl acetylenedicarboxylate adduct ofbenzoporphyrin, δ-aminolevulinic acid, benzonaphthoporphyrazine,naturally occurring porphyrin, ALA-induced protoporphyrin IX, syntheticdichlorin, bacteriochlorin tetra(hydroxyphenyl) porphyrin, purpurin,octaethylpurpurin derivative, etiopurpurin, tin-etio-purpurin,porphycene, chlorin, chlorin e₆, mono-1-aspartyl derivative of chlorine₆, di-1-aspartyl derivative of chlorin e₆, tin(IV) chlorin e₆,meta-tetrahydroxyphenylchlorin, chlorin e₆ monoethylendiamine monamide,verdin, zinc methyl pyroverdin, copro II verdin trimethyl ester,deuteroverdin methyl ester, pheophorbide derivative, pyropheophorbide,texaphyrin, lutetium (III) texaphyrin, or gadolinium(III) texaphyrin.

In another aspect, on or both of the photosensitizers, X and X′, can bea photoactive dye, including merocyanine, phthalocyanine, chloroaluminumphthalocyanine, sulfonated aluminum PC, ring-substituted cationic PC,sulfonated AlPc, disulfonated or tetrasulfonated derivative, sulfonatedaluminum naphthalocyanine, naphthalocyanine, tetracyanoethylene adduct,crystal violet, azure β chloride, benzophenothiazinium,benzophenothiazinium chloride (EtNBS), phenothiazine derivative, roseBengal, toluidine blue derviatives, toluidine blue O (TBO); methyleneblue (MB), new methylene blue N (NMMB), new methylene blue BB, newmethylene blue FR, 1,9-dimethylmethylene blue chloride (DMMB), methyleneblue derivatives, methylene green, methylene violet Bernthsen, methyleneviolet 3RAX, Nile blue, Nile blue derivatives, malachite green, Azureblue A, Azure blue B, Azure blue C, safranine O, neutral red,5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,thiopyronine, or thionine.

In a particular aspect, X and X′ of formula I can bebenzophenothiazinium chloride (EtNBS) and the linker (L) can be acepahlosporin or a fragment thereof.

In yet another aspect, formula I can include a targeting moiety, whichtargets a cell or tissue, such as a pathogen or a host cell infectedwith a pathogen, or a macrophage, or a cell or tissue or bodilycomponent, such as a liposome or peptide.

Formula I may further include a binder effective to quenchphotoactivation, such as a fluorophore or another photosenstitizer.

In addition, certain aspects provide formula I with a backbone, whichcan be utilized to couple one or more photosensitizers, binderseffective to quench photoactivation, and targeting moieties.

In another aspect, the present invention provides a method for detectinga beta-lactamase activity in a sample, comprising the steps of:contacting the sample with a photosensitizer composition comprising aplurality of photosensitizers that are conjugated to a linker, whereinthe linker comprise a cleavage site for a beta-lactamase and wherein thephotosensitizers are in a quenched state; cleaving the linker todequench the photosensitizers; light-activating the composition toproduce fluorescence signal; and detecting the fluorescence signal witha detector, thereby detecting a beta-lactamase activity.

The sample can be a biological sample isolated from an infection in asubject, such as from a mammal or human. In one aspect, the infection iscaused by an antibiotic-resistant pathogen, such as any of those listedabove.

The the method for detecting a beta-lactamase activity in a sample canalso include the step of quantifying the detected fluorescence signal.

The present invention also provides a method for typing beta-lactamaseactivity in a sample, comprising: performing a non-competitive reactioncomprising the steps of (a) contacting the sample with a photosensitizercomposition comprising a plurality of photosensitizers that areconjugated to a linker, wherein the linker comprise a cleavage site fora beta-lactamase and wherein the photosensitizers are in a quenchedstate; (b) cleaving the linker to dequench the photosensitizers; (c)light-activating the composition to produce a fluorescence signal; and(d) quantifying the fluorescence signal with a detector.

The present invention also provides a method for determining anappropriate antibiotic regimen to be administered to a subject in needthereof, comprising the steps of: typing a beta-lactamase activity of asample according to the invention based on one or more competingbeta-lactam substrates; and administering to the subject in need thereofan antibiotic regimen that excludes any antibiotic having a structurethat is the same or is similar to the competing beta-lactam substratesthat are shown to be cleavable by the beta-lactamase activity of thesample.

Methods of typing beta-lactamases and for detecting and/or quantitatingbeta-lactamase activities can be done on any suitable sample containinga beta-lactamase enzyme. In one aspect, the sample is obtained frominfected cells or tissues of a subject having a bacterial infection,such as an infection by a Gram (−) or Gram (+) bacterial pathogen.Sample cells and/or tissues can be infected with or containStaphylococcus, Enterococcus, Enterobacter, Escherichia, Haemophilus,Neisseria, Klebsiella, Pasteurella, Proteus, Pseudomonas,Streptophomonas, Burkholderia, Acinetobacter, Serratia, and Salmonellaspp., as well as more particularly Staphylococcus aureus, Staphylococcusepidermis, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Haemophilus influenzae, Neisseria gonorrhea, Klebsiellapneumoniae, Pasteurella multocida, Proteus mirabilis, Pseudomonasaeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia,Acinetobacter baumannii, Enterobacter aerogines, Enterobacter cloacae,Serratia marcescens, Salmonella enterica, and Salmonella typhimurium.

The present invention also includes a kit for detecting a beta-lactamaseactivity in a sample containing a photosensitizer composition describedherein and instructions for using the photosensitizer composition todetect a beta-lactamase activity in a sample.

The instant invention further provides a kit for typing a beta-lactamaseactivity in a sample comprising a photosensitizer composition describedherein and instructions for using the photosensitizer composition totype a beta-lactamase activity in a sample.

Other aspects of the invention are described in the followingdisclosure, and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments aspectsdescribed, may be understood in conjunction with the accompanyingdrawings, which incorporated herein by reference. Various features andaspects of the present invention will now be described by way ofnon-limiting examples and with reference to the accompanying drawings,in which:

FIG. 1 schematically depicts the development of a carbamate-linkedphotosensitizer (PS) that is inactive (with or without light) whilelinked and is light-activatable only when released by the β-lactamaseenzyme-mediated cleavage.

FIG. 2A shows ¹H NMR spectra obtained for 7-[(2-phenylacetyl)amino]cephalosporanic acid in CDCl₃ as a solvent.

FIG. 2B shows ¹H NMR spectrum obtained for 7-[(2-phenylacetyl)amino]3-hydrodxymethy cephalosporanic acid in DMSO-d₆ as a solvent. Majorproton peaks are marked on the spectra.

FIGS. 3A-B show MS spectra obtained for (a) 7-[(2-phenylacetyl)amino]3-hydrodxymethy cephalosporanic acid; and (b) cephalosporanicacid-toluidine blue O prodrug.

FIG. 4 shows UV-visible spectra obtained for the photosensitizer (TBO)vs. the cephalosporanic acid-photosensitizer prodrug in ethanol at aconcentration of 2.0×10⁻⁵ M.

FIG. 5 shows fluorescence emission spectra obtained for thephotosensitizer (TBO) vs. the cephalosporanic acid-photosensitizerprodrug in ethanol at 635 nm excitation.

FIGS. 6A-B show plots of (a) fluorescence emission vs. wavelength and(b) flurorescence emission vs. time for the cephalosporanicacid-photosensitizer prodrug, depicting the enzyme-mediated cleavage ofthe prodrug.

FIG. 7 depicts the specific mechanism for bacterial beta-lactamaseenzyme mediated photosensitizer prodrug (β-LEAPP) activation indemonstrating the principle of the use of hydrolytic bacterial virulenceenzymes for the specific release of active photosensitizer from aquenched state.

FIG. 8 depicts the synthesis of β-LEAPP.

FIG. 9 shows the fluorescence emission of β-LEAPP incubated withdifferent concentrations of B. cereus Penicillinase expressed as afunction of time. The values listed in the chart legend are theconcentrations of Penicillinase in units of enzyme per milliliter. Datais representative of three experimental repeats.

FIG. 10 shows the double reciprocal plot of the instantaneous velocityof the increase in β-LEAPP fluorescence as a function of theconcentration of B. cereus Penicillinase. The instantaneous velocity wasdetermined over the range of the first 20 readings taken over the first20 minutes. Data consists of experimental repeats.

FIGS. 11A-B show the fluorescence emission of β-LEAPP produced inincubation with various strains of bacteria as a function of time. GraphA shows ATCC 29213 S. aureus beta-lactamase non-producer, MRSA 8150,8179, 9307 (all clinical isolates and beta-lactamase producers), and ano-cell control. Graph B shows ATCC 25922 E. coli beta-lactamasenon-producer, ATCC BAA196 E. coli ESBL producer, a no-cell control.

FIG. 12 shows a suitable multiwell optically transparent plate i.e. 96well culture plate which be used according to this examples describedherein to carry out the reactions to determine enzyme substratespecificity.

FIGS. 13A-D show a Comparison of Inhibition Constants for Pureβ-Lactamase & Bacterial Suspensions, with Inhibition Profiles & MinimumInhibitory Concentrations of Bacillus cereus 5/β. (a.) inhibitionconstants of a panel of β-lactams for the competitive substrateinhibition of β-LEAP hydrolysis by B. cereus β-lactamase, (b.)inhibition constants for β-LEAP hydrolysis by bacterial suspensions,(c.) inhibition profiles of panel of β-lactams for B. cereus, (d.) MICsof panel of β-lactams for B. cereus. Amoxicillin, clavulanic acid andampicillin did not inhibit the growth of B. cereus and were not includedin (c.) and (d.). Statistical significance was determined via Tukeymultiple comparison test using Graphpad Prism 5. Horizontal barsconnected by solid intersections indicate significant difference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to photosensitizer compositions, and tomethods and kits utilizing the photosensitizer compositions for thedetection, quantitation and characterization of beta-lactamase enzymesfor purposes of clinical research and diagnostics. The photosensitizercompositions of the invention comprise substrates for beta-lactamases aslinkers for a plurality of photosensitizer molecules. Prior to cleavageof the linker moiety, the plurality of photosensitizer molecules arequenched such that they are not in a activatable state. When thebeta-lactamase cleavage sight on the linker is cleaved, thephotosensitizer molecules become physically separated, at which time,they become activatable and may produce a signal (e.g., fluorescence)concomitant with activation (e.g., light-activation). This feature canbe utilized in accordance with the invention in a variety of methods,including quantitative detection of beta-lactamase activity in varioussamples, determination of the substrate specificity of differentbeta-lactamase enzymes, and the characterization of beta-lactamantibiotic resistant pathogens for selection of appropriateantimicrobial therapies.

It will be appreciated that determining and initiating the appropriatecourse of antibiotics in an expedient manner is critical to patientoutcome as well as to the reduction of the frequency of occurrence ofantibiotic resistant bacterial infections, especially in the case ofbeta-lactamase producing organisms. Current protocols for the clinical,definitive identification of a beta-lactmase phenotype and/or genotyperequires from about 1 to 3 days of culturing with in vitrosusceptibility testing and/or polymerase chain reaction (PCR) relatedtechniques, respectively, for the detection of beta-lactamase activity.These methods can be misleading and are time consuming.

In part to answer the deficiencies known in the art relating to methodsfor detecting beta-lactamase activity, the present invention providesnew methods for detecting and evaluating beta-lactamase activity whichare more sensitive and rapid and easier to perform, and which reliablyand expediently provide both qualitative and quantitative information asto an enzyme's activity and/or substrate specificity.

Definitions

The term “beta-lactam,” or alternatively, “β-lactam” or “β-lactammoiety,” and the like, refers to the four-atom beta-lactam ringstructure characteristic of the cleavage site of substrates ofbeta-lactamases, such as penicillins, cephalosporins, cephamycins andcarbapenems. A beta-lactam ring has the following structure:

A “beta-lactam derivative,” as used herein, refers to any compoundresembling a beta-lactam ring moiety that is capable of being cleaved bya beta-lactamase enzyme or catalytic fragment thereof. A beta-lactamderivative may be derived from a beta-lactam through chemicalmodification or may be independently synthesized.

As used herein, the term “beta-lactamase” or alternatively,“β-lactamase” denotes a protein capable of catalyzing cleavage of abeta-lactamase substrate, such as, a beta-lactam containing molecule(e.g., a beta-lactam antibiotic) or fragments or derivative thereof.Beta-lactamases of the invention can be obtained from any natural (e.g.,infectious tissue or isolated pathogenic strain), commercial, oracademic source. Beta-lactamases are generally known in the art and arefurther described in Waley, The Chemistry of beta-Lactamase, Page Ed.,Chapman & Hall, London, (1992) 198-228 (the contents of which areincorporated herein by reference).

Fragments and/or derivatives of beta-lactamases and their preparationare known in the art. One skilled in the art will recognize thatindividual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservative substitutions” or “conservatively modifiedvariations” where the alterations result in the substitution of an aminoacid with a chemically similar amino acid. Conservative substitutiontables providing functionally similar amino acids are well known in theart. The following five groups each contain amino acids that areexemplary conservative substitutions for one another:

Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine(I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R),Lysine (K), Histidine (H); and

Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine(Q). See also, Creighton, Proteins, W.H. Freeman and Company, 1984, foradditional groupings of amino acid substitutions.

In addition, individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids in an encoded sequence are also “conservative variations.”Variants of a peptide are typically characterized by possession of atleast about 50% sequence identity counted over the full length alignmentwith the amino acid sequence of the peptide using the NCBI Blast 2.0,gapped blastp set to default parameters, more preferably about 60% or70%, even more preferably about 80% or 90%, or even 95% or 99% sequenceidentity.

The term “photosensitizer” refers to an activatable compound thatproduces a signal when light activated. The photosensitizers of theinvention can produce a photochemical or phototoxic effect on a cellwhen light activated, i.e., produce a reactive species when lightactivated. The photosensitizers of the invention can includephotosensitizer fragments and/or derivatives of known photosensitizers,which have the same or substantially the same function as the knownphotosensitizers, which means that function which is at least about 50%of the function of an original photosensitizer, more preferably about60% or 70%, or still more preferably about 80% or 90%, or even morepreferably about 95% or 99% the function of the known photosensitizercompound.

The photosensitizers of the invention can include “photoactive dyes,”which, as used herein, refers to those photosensitizers that produce afluorescent signal when activated, but not necessarily a reactivespecies in phototoxic amounts (i.e., a phototoxic species). Signals thatcan be measured from a photoactive dye include: (i) phosphorescence,(ii) fluorescence, (iii) reactive molecular species. The first two are acomponent of light itself and the last one is a physic-chemicalconsequence of light absorption by the photoactive dye. The photoactivedyes of the invention may also be fragments and/or derivatives of aknown photoactive dyes which have the same or substantially the samefunction as a known photoactive dye, which means a function that is atleast about 50% of the function of a known photoactive dye, morepreferably about 60% or 70%, or still more preferably about 80% or 90%,or even more preferably about 95% or 99% the function of a knownphotoactive dye.

Depending on the wavelength and power of light administered, aphotosensitizer can be activated to fluoresce and, therefore, act as aphotoactive dye, but not produce a phototoxic species. The wavelengthand power of light can be adapted by methods known to those skilled inthe art to bring about a phototoxic effect where desired.

The term “photosensitizer composition,” as used herein, refers tochemical constructs having one or more photosensitizers (or fragmentsand/or derivatives thereof), as well as other materials, such aslinkers, backbones, targeting moieties and binders, that may be couplethereto.

As used herein, the term “fluorescent dye” refers to dyes that arefluorescent when illuminated with light but do not produce reactivespecies that are phototoxic.

Any compound or moiety of the invention that is fluorescent in one ormore states can contain one or more “fluorophores,” which refers to acompound or portion thereof which exhibits fluorescence. The term“fluorogenic” refers to a compound or composition that becomesfluorescent or demonstrates a change in its fluorescence (such as anincrease or decrease in fluorescence intensity or a change in itsfluorescence spectrum) upon interacting with another substance, forexample, upon binding to a biological compound or metal ion, uponreaction with another molecule or upon metabolism by an enzyme.Fluorophores may be substituted to alter their solubility, spectralproperties and/or physical properties. Numerous fluorophores andfluorogenic compounds and compositions are known to those skilled in theart and include, but are not limited to, benzofurans, quinolines,quinazolines, quinazolinones, indoles, benzazoles, indodicarbocyanines,borapolyazaindacenes and xanthenes, with the latter includingfluoresceins, rhodamines and rhodols as well as other fluorophoresdescribed in Haugland, Molecular Probes, Inc. Handbook of FluorescentProbes and Research Chemicals, (9^(th) ed., including the CD-ROM,September 2002), and include the photosensitizers, photoactive dyes, andfluorescent compounds and moieties of the invention.

The term “conjugated,” as used herein, refers to the coupling orassociation of two or more molecules (e.g., a photosensitizer and alinker), usually by covalently bonding.

As used herein, the term “detectable” or “directly detectable,” or thelike, refers to the presence of a detectable signal generated from acompound of the invention, e.g., a photosensitizer, that is detectableby observation, instrumentation, or film without requiring chemicalmodifications or additional substances.

As used herein, the term “linker” refers to an agent capable of linkingtwo or three components (e.g., compounds or moieties) of thephotosensitizer composition together (e.g., a photosensitizer to anotherphotosensitizer, a photosensitizer to a binder, a photosensitizer to abackbone, a binder to a backbone, a photosensitizer to a targetingmoiety, or a binder to a targeting moiety). A linker is a relativelysmall moiety with only two or three conjugatable end groups to linkbetween two or three molecules/targeting agents. The “linker” may becleavable. Examples of linkers are described herein.

In addition to enzymatically cleavable groups, e.g., beta-lactammoieties, it is within the scope of the present invention to include oneor more sites in a linker that are cleaved by the action of an agentother than an enzyme. Exemplary non-enzymatic cleavage agents include,but are not limited to, acids, bases, light (e.g., nitrobenzylderivatives, phenacyl groups, benzoin esters), and heat. Many cleaveablegroups are known in the art. See, for example, Jung et al., Biochem.Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265:14518-14525 (1990); Zarling et al., J. Immunol, 124: 913-920 (1980);Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J.Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol., 143:1859-1867 (1989), each of which are incorporated by reference.

As used herein, the term “backbone” refers to an agent that functions tocouple one or more components of a photosensitizer composition of theinvention, such as, for example, a polyamino acid or like agent that islinked to one or more photosensitizers and/or one or more binders and/orone or more targeting moieties The backbone itself additionally can be atargeting moiety, e.g. polylysine. A “backbone” as used herein is as amoiety higher in molecular weight and capable of loading morephotoactive molecules than a ‘linker’. Backbone can be a polymericstructure which provides a base to add multiple units (more than three).Examples of backbones that can be used according to the invention,include, but are not limited to polyethylene glycol and polyproline.

The term “sample,” as used herein, refers to any material that maycontain a beta-lactamase or a nucleotide sequence coding for abeta-lactamase, or to a material to which a beta-lactamase or anucleotide sequence coding for a beta-lactamase is added. Typically, thesample may be obtained from and/or contain a bodily tissue, cell, orfluid of a subject, and can comprise endogenous host cell proteins,nucleic acid polymers, nucleotides, oligonucleotides proteins orpeptides isolated or obtained therefrom. The sample may be presented inany suitable physical form, such as, for example, a fluid dispersion,such as an aqueous solution, a cell culture (viable or otherwise) orimmobilized on a solid or semi solid surface such as a polyacrylamidegel, membrane blot or microarray.

As used herein, the term “binder” refers to an agent that absorbs energyfrom an adjacent, activated photosensitizer or otherwise inactivates thephotosensitizer, and, thus, quenches the photosensitizer. A “binder” maybe used synonymously with the term “quencher,” which may also refer to acompound or moiety that absorbs energy from an excited donor compound ormoiety. A quencher may absorb the fluorescent photons emitted by a donorcompound, thereby masking the donor compound's fluroscence. A binder canparticipate in both static (ground state) and dynamic (excited state)quenching. That is, in the context of an EtNBS-linker-EtNBSconfiguration (an example of static quenching), either of the EtNBSmoieties can be considered to be the binder, while the other EtNBS canbe considered as the fluorophore. Alternatively, the inventioncontemplates an embodiment of a photoactivatable prodrug as disclosedherein where the prodrug has the configuration EtNBS-linker-binder. Thisconfiguration would represent an example of dynamic-type quenching inwhich the binder (e.g., fluoroscein) functions to quench the signalgenerated by EtNBS until the two moieties are separated by cleavage ofthe linker.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are,unless specified otherwise, used interchangeably. Peptides,polypeptides, and proteins used in methods and compositions describedherein can be recombinant, purified from natural sources, or chemicallysynthesized. For example, reference to the use of a bacterial protein ora protein from bacteria, includes the use of recombinantly producedmolecules, molecules purified from natural sources, or chemicallysynthesized molecules.

The term “subject” is used herein to refer to a living animal, includinga human. The subject can be infected with or carrying an unwantedorganism, e.g., a bacterial infection.

As used herein, “pathogen” or “target organism” means an organism whichcauses or aggravates a disorder, such as an infection, granuloma, orother adverse immune response.

The term “obtaining,” as in “obtaining” the “photosensitizercomposition,” “linker” or “binder,” is intended to include purchasing,synthesizing or otherwise acquiring the elements of the invention.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure.

Antibiotics, Resistance and Beta-Lactamase

While not intending to limit the present invention, the followinggeneral description of antibiotics, resistance and beta-lactamase isprovided.

In general, antimicrobial chemotherapeutics are classified based onchemical structure and mode of action. In particular, antibiotics can beclassified as (a) agents that inhibit the synthesis of bacterial cellwalls, including the β-lactam class of antibiotics (e.g., penicillins,cephalosporins, or carbapenems) or other dissimilar agents such asvancomycin and bacitracin, (b) agents that act to permeabilize thecellular membrane causing a toxic release of intracellular material(e.g., detergents such as polymyxin), (c) agents that disrupt thefunction of 30S or 50S ribosomal subunits to interrupt protein synthesis(e.g., chloramphenicol, tetracyclines, or erythromycine), (d) agentsthat inhibit or block bacterial nucleic acid synthesis or metabolism(e.g., rifampin, rifabutin, or quinolones), and (e) agents that block orinhibit bacterial metabolism (e.g., trimethoprim or sulfonamides).Regarding beta-lactam antibiotics, in particular, resistance to suchcompounds is due mainly to the ability of resistant bacteria to expressbeta-lactamase enzymes, which are released from the cell and whichhydrolyze the beta-lactam rings of the antibiotics, thereby inactivatingthem.

The β-lactam class of antibiotics represent a large and important classof antibiotics whose overall effectiveness is threatened by theemergence of resistance, which is in particular, caused by the continuedappearance of beta-lactamase enzymes in various bacteria.Beta-lactamases represent an efficient mechanism devised by bacteria toescape the lethal action of beta-lactam antibiotics. They can bechromosomal or plasmid encoded, produced in a constitutive or induciblemanner, and can be secreted into the periplasmic space of Gram negativestrains or into the outer medium by their Gram positive counterparts.The ubiquitous occurrence of beta-lactamases in bacteria and theirassociation with clinical resistance has sustained a strong interest inthese enzymes. Just a few years after the clinical debut of penicillinin 1944, the first reports of resistance to the beta-lactam antibiotischad emerged. Currently, there are over 500 known differentbeta-lactamases, of which 200 of them areextended-spectrum-beta-lactamases (ESBLs) (Paterson et al., Clin.Microbio. Rev. 2005, 18:657-686).

As briefly mentioned above, β-lactamases are organized under oneclassification scheme into four molecular classes (A, B, C and D) basedon their amino acid sequences. Class A enzymes have a molecular weightof about 29 kDa and preferentially hydrolyze penicillins. Examples ofclass A enzymes include the β-lactamase of Staphylococcus aureus. ClassB enzymes include metalloenzymes that have a broader substrate profilethan the other classes of β-lactamases. Class C enzymes have molecularweights of approximately 39 kDa and include the chromosomalcephalosporinases of gram-negative bacteria, which are responsible forthe resistance of gram-negative bacteria to a variety of bothtraditional and newly designed antibiotics. In addition, class C enzymesalso include the lactamase of P99 Enterobacter cloacae, which isresponsible for making this Enterobacter species one of the most widelyspread bacterial agents in United States hospitals. The recentlyrecognized class D enzymes are serine hydrolases, which exhibit a uniquesubstrate profile.

The four classes of A, B, C and D beta-lactamases can be furthercategorized into functional clusters through the Bush-Jacoby-Medeirosclassification system (Philippon et al., 1998). In considering two ofthe four classes, A and C, one can demonstrate that there is a highlevel of ambiguity in the relationship between the primary amino acidsequence of the enzymes and the functional phenotypes that theirexpression confers. The class C beta-lactamases include both chromosomaland plasmid borne members whose activity is highly similar, withconserved motifs, but whose total sequence identity can vary as much as40% (Philippon et al., 1998). The functional phenotype associated withtheir expression is characterized by resistance to amino penicillins,first-generation cephalosporins, cefoxitin, and toamoxicillin/clavulanate combinatorial therapy (Philippon et al., 1998).Conversely, the class A beta-lactamases, encompassing the ESBLs, canshare a high degree of sequence identity, but are the most functionallydiverse of the four Amber classes. The range of phenotypes within classA includes resistance to penicillins only, to penicillins and somecephalosporins, to cephalosporins and cefuroxime, to third generationcephalosporins, to beta-lactamase inactivators, or to imipenem(Philippon et al., 1998). Some class A enzymes, although 97% similar inprimary structure, exhibit great differences in kinetics for varioussubstrates. This relationship is exemplified by the enzymes TEM1 andTEM12 (ESBL) where a single amino acid difference in a primary sequenceof 286 amino acids changes the resistance phenotype from that ofresistance to broad-spectrum cephalosporins to that of resistance tothird-generation extended spectrum cephalosporins (Philippon et al.,1998).

Other details regarding the nature of antibiotics, resistance mechanismsand beta-lactamase enzymes will be well appreciated by those havingordinary skill in the art.

Compositions of the Invention Photosensitizer Compositions of theInvention

The present invention provides, in one aspect, a photosensitizercomposition comprising one or more photosensitizers that are conjugatedto a linker, wherein the linker comprises a cleavage site (e.g., abeta-lactamase enzyme cleavage site) and wherein the one or morephotosensitizers, when coupled together by a linker, are in a quenchedstate. Upon cleavage of the linker (e.g., by an enzyme capable ofcleaving the cleavage site), the one or more photosensitizers becomeunquenched and photoactivatable. The unquenched photosensitizers canthen be activated to produce a signal (e.g., light emission), which canthen be detected by a detection means (e.g., a fluorimeter). The linkerof the invention can comprise one or more beta-lactamase cleavage sites,each site comprising at least one beta-lactam moiety or functionalderivative thereof.

Any suitable physical arrangement of the one or more photosensitizersand the linker is contemplated by the present invention, so long as thecleavage of the linker results in, either directly or indirectly, theunquenching of the one or more photosensitizers. It will be appreciatedby those having ordinary skill in the art that quenching is a reductionin intensity of the excited state of a photosensitizer. Quenching cancan be attained in many different ways. For example, two mechanismsinclude: (i) static quenching caused by dimerization in ground state and(ii) dynamic quenching caused by FRET (fluorescence resonance energytransfer), that represents energy transfer between an excited donor anda ground-state acceptor molecule. Quenching may also be attained byother methods, including, for example, photoinduced electron transfer,paramagnetic enhancement of intersystem crossing, Dexter exchangecoupling, and exciton coupling such as the formation of dark complexes.Quenched state depends on the distance between two photoactivemolecules. This distance can be defined by the choice of linkermolecule. For example, a flexible linker can be used to alter thedistance between two molecules with respect to temperature and solventconditions and analyze the best distance for quenching. Otherwise, arigid linker can be employed to observe quenching efficiency at a fixeddistance. Quenching refers to any process that decreases thefluorescence intensity of a sample and includes excited-state reactions,molecular rearrangements, energy transfer, ground-state complexformation, and collisional quenching.

In a particular aspect, the photosensitizer composition of the presentinvention is represented by the general formula I:

X-L-X′,

wherein at least one of X or X′ is a photosensitizer in accordance withthe invention and wherein L represents an uncleaved linker. In anotheraspect, X and X′ can be the same or different photosensitizers. In yetanother aspect, X can be a photosensitizer and X′ can be a binder orquencher which quenches the photosensitizer. In certain other aspects, Xand/or X′ can be a photoactive dye, such as, for example, a penothiazine(e.g., benzophenothiazinium chloride (EtNBS)) or functional derivativeor fragment thereof. The linker (L) can be any suitable linker disclosedherein, which at a minimum contains at least one enzyme-cleavable site(e.g., a beta-lactam moiety).

In a particular aspect, X and X′ are each benzophenothiazinium chloride(EtNBS) and L comprises a beta-lactam moiety or derivative thereof, suchas, for example a penicillin or a cephalosporin, or derivative thereof.X and X′ can be benzophenothiazinium chloride (EtNBS) and L can comprisea cephalosporin.

In other aspects, the photosensitizer compositions of the invention caninclude one or more targeting moieties, which can be coupled to thelinker (L) or to one or more of the photosensitizers X or X′. Thephotosensitizer compositions in other embodiments can also be coupled toa backbone. Targeting moieties useful in the present invention includeantibodies, aptamers, proteins, and peptides. Targeting moieties includebiological macromolecules; proteins, nucleic acids, lipids, andcarbohydrates, as well as small molecules and chemical functionalgroups.

The following description provides further guidance regarding the natureof the components of the photosensitizer compositions of the invention.

Photosensitizers

Any suitable photosensitizer or combination of photosensitizers can bejoined to any suitable linker to form the photosensitizer compositionsof the invention, provided that the photo sensitizers are quenched whenthe linker is uncleaved, but become unquenched when the linker iscleaved. Photosensitizers of the invention can have the followinggeneral characterisitics: Reasonable fluorescence quantum yield fordiagnostics, significant phototoxicity for therapy, minimum darktoxicity, absorption in light region of spectrum (400-800 nm), and aFortser radius between FRET pairs of 2-8 nm.

The photosensitizers of the invention can be amphiphilic, meaning thatthey share the opposing properties of being water-soluble, yethydrophobic. The photosensitizer should be water-soluble in order topass through the bloodstream systemically, however, it should also behydrophobic enough to pass across cell membranes. Modifications, such asattaching polar residues (amino acids, sugars, and nucleosides) to thehydrophobic porphyrin ring, can alter polarity and partitioncoefficients to desired levels. Such methods of modification are wellknown in the art.

In specific embodiments, photosensitizers of the present inventionabsorb light at a relatively long wavelength, thereby absorbing at lowenergy. Low-energy light can travel further through tissue thanhigh-energy light, which becomes scattered. Optimal tissue penetrationby light occurs between about 650 and about 800 nm. Porphyrins found inred blood cells typically absorb at about 630 nm, and new, modifiedporphyrins have optical spectra that have been “red-shifted”, in otherwords, absorbs lower energy light. Other naturally occurring compoundshave optical spectra that is red-shifted with respect to porphyrin, suchas chlorins found in chlorophyll (about 640 to about 670 nm) orbacteriochlorins found in photosynthetic bacteria (about 750 to about820 nm).

In certain embodiments, the photosensitizers of the invention caninclude porphyrins and/or hydroporphyrins. Porphyrins andhydroporphyrins can include, but are not limited to, Photofrin® RTM(porfimer sodium), hematoporphyrin IX, hematoporphyrin esters,dihematoporphyrin ester, synthetic diporphyrins, 0-substitutedtetraphenyl porphyrins (picket fence porphyrins), 3,1-meso tetrakis(o-propionamido phenyl) porphyrin, hydroporphyrins, benzoporphyrinderivatives, benzoporphyrin monoacid derivatives (BPD-MA), monoacid ring“a” derivatives, tetracyanoethylene adducts of benzoporphyrin, dimethylacetylenedicarboxylate adducts of benzoporphyrin, endogenous metabolicprecursors, 6-aminolevulinic acid, benzonaphthoporphyrazines, naturallyoccurring porphyrins, ALA-induced protoporphyrin IX, syntheticdichlorins, bacteriochlorins of the tetra(hydroxyphenyl) porphyrinseries, purpurins, tin and zinc derivatives of octaethylpurpurin,etiopurpurin, tin-etio-purpurin, porphycenes, chlorins, chlorin e₆,mono-1-aspartyl derivative of chlorin e₆, di-1-aspartyl derivative ofchlorin e₆, tin(IV) chlorin e₆, meta-tetrahydroxyphenylchlorin, chlorine₆ monoethylendiamine monamide, verdins such as, but not limited to zincmethyl pyroverdin (ZNMPV), copro II verdin trimethyl ester (CVTME) anddeuteroverdin methyl ester (DVME), pheophorbide derivatives, andpyropheophorbide compounds, texaphyrins with or without substitutedlanthanides or metals, lutetium (III) texaphyrin, and gadolinium(III)texaphyrin, or a functional derivative or fragment thereof, i.e.,compounds that are chemically similar and/or are small portions of theoriginal compound which perform the same or substantially the samefunction. Where any moiety, fragment or derivative of a compound of theinvention performs “substantially the same function” as the originalcompound, that moiety, fragment or derivative performs at least about50% of the activity of the original compound, more preferably about 60%or 70%, or still more preferably about 80% or 90%, or even morepreferably about 95% or 99% the activity of the original compound.

Porphyrins, hydroporphyrins, benzoporphyrins, and derivatives are allrelated in structure to hematoporphyrin, a molecule that is abiosynthetic precursor of heme, which is the primary constituent ofhemoglobin, found in erythrocytes. First-generation and naturallyoccurring porphyrins are excited at about 630 nm and have an overall lowfluorescent quantum yield and low efficiency in generating reactiveoxygen species. Light at about 630 nm can only penetrate tissues to adepth of about 3 mm, however there are derivatives that have been‘red-shifted’ to absorb at longer wavelengths, such as thebenzoporphyrins BPD-MA (Verteporfin). Thus, these ‘red-shifted’derivatives show less collateral toxicity compared to first-generationporphyrins.

Porphyrin derivatives also include chlorins and bacteriochlorins,however these have the unique property of hydrogenated exo-pyrroledouble bonds on the porphyrin ring backbone, which allows for absorptionat wavelengths greater than about 650 nm. Chlorins are derived fromchlorophyll, and modified chlorins such as meta-tetrahydroxyphenylchlorin (mTHPC) have functional groups to increasesolubility. Bacteriochlorins are derived from photosynthetic bacteriaand are further red-shifted to about 740 nm. A specific embodiment ofthe invention uses chlorin_(e6).

Porphryin derivatives also include purpurins, porphycenes, and verdins,which have efficacies similar to or exceeding hematoporphyrin. Purpurinscontain the basic porphyrin macrocycle, but are red-shifted to about 715nm. Porphycenes have similar activation wavelengths to hematoporphyrin(about 635 nm), but have higher fluorescence quantum yields. Verdinscontain a cyclohexanone ring fused to one of the pyrroles of theporphyrin ring. Phorbides and pheophorbides are derived fromchlorophylls and have 20 times the effectiveness of hematoporphyrin.Texaphyrins are new metal-coordinating expanded porphyrins. The uniquefeature of texaphyrins is the presence of five, instead of four,coordinating nitrogens within the pyrrole rings. This allows forcoordination of larger metal cations, such as trivalent lanthanides.Gadolinium and lutetium are used as the coordinating metals. In aspecific embodiment, the photosensitizer can be Antrin®, otherwise knownas motexafin lutetium.

5-aminolevulinic acid (ALA) is a precursor in the heme biosyntheticpathway, and exogenous administration of this compound causes a shift inequilibrium of downstream reactions in the pathway. In other words, theformation of the immediate precursor to heme, protoporphyrin IX, isdependent on the rate of 5-aminolevulinic acid synthesis, governed in anegative-feedback manner by concentration of free heme. Conversion ofprotoporphyrin DC is slow, and where desired, administration ofexogenous ALA can bypass the negative-feedback mechanism and result inaccumulation of phototoxic levels of ALA-induced protoporphyrin IX. ALAis rapidly cleared from the body, but like hematoporphyrin, has anabsorption wavelength of about 630 nm.

First-generation photosensitizers are exemplified by the porphyrinderivative Photofrin®, also known as porfimer sodium. Photofrin® isderived from hematoporphyrin-IX by acid treatment and has been approvedby the Food and Drug Administration for use in PDT. Photofrin® ischaracterized as a complex and inseparable mixture of monomers, dimers,and higher oligomers. There has been substantial effort in the field todevelop pure substances that can be used as successful photosensitizers.Thus, in a specific embodiment, the photosensitizer is a benzoporphyrinderivative (“BPD”), such as BPD-MA, also commercially known asVerteporfin. U.S. Pat. No. 4,883,790 describes BPDs. Verteporfin hasbeen thoroughly characterized (Richter et al., 1987; Aveline et al.,1994; Levy, 1994) and it has been found to be a highly potentphotosensitizer for PDT. Verteporfin has been used in PDT treatment ofcertain types of macular degeneration, and is thought to specificallytarget sites of new blood vessel growth, or angiogenesis, such as thoseobserved in “wet” macular degeneration. Verteporfin is typicallyadministered intravenously, with an optimal incubation time range from1.5 to 6 hours. Verteporfin absorbs at 690 nm, and is activated withcommonly available light sources. One tetrapyrrole-based photosensitizerhaving recent success in the clinic is MV0633 (Miravant).

The photosensitizers can have a chemical structure that includesmultiple conjugated rings that allow for light absorption andphotoactivation. Such specific compounds include motexafin lutetium(Antrin®) and chlorin_(e6).

The photosensitizers of the present invention also include cyanines andother photoactive dyes. Cyanine and other dyes include but are notlimited to a merocyanine, phthalocyanine, chloroaluminum phthalocyanine,sulfonated aluminum PC, ring-substituted cationic PC, sulfonated AlPc,disulfonated or tetrasulfonated derivative, sulfonated aluminumnaphthalocyanine, naphthalocyanine, tetracyanoethylene adduct, crystalviolet, azure β chloride, benzophenothiazinium, benzophenothiaziniumchloride (EtNBS), phenothiazine derivative, phenothiaziniums such asrose Bengal, toluidine blue derviatives, toluidine blue O (TBO),methylene blue (MB), new methylene blue N (NMMB), new methylene blue BB,new methylene blue FR, 1,9-dimethylmethylene blue chloride (DMMB),methylene blue derivatives, methylene green, methylene violet Bernthsen,methylene violet 3RAX, Nile blue, Nile blue derivatives, malachitegreen, Azure blue A, Azure blue B, Azure blue C, safranine O, neutralred, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,thiopyronine, or thionine.

Cyanines are deep blue or purple compounds that are similar in structureto porphyrins. However, these dyes are much more stable to heat, light,and strong acids and bases than porphyrin molecules. Cyanines,phthalocyanines, and naphthalocyanines are chemically pure compoundsthat absorb light of longer wavelengths than hematoporphyrin derivativeswith absorption maxima at about 680 nm. Phthalocyanines, belonging to anew generation of substances for PDT are chelated with a variety ofdiamagnetic metals, chiefly aluminum and zinc, which enhance theirphototoxicity. A ring substitution of the phthalocyanines withsulfonated groups will increase solubility and affect the cellularuptake. Less sulfonated compounds, which are more lipophilic, show thebest membrane-penetrating properties and highest biological activity.The kinetics are much more rapid than those of HPD, where, for example,high tumor to tissue ratios (8:1) were observed after 1-3 hours. Thecyanines are eliminated rapidly and almost no fluorescence can be seenin the tissue of interest after 24 hours.

Other photoactive dyes such as methylene blue and rose bengal, are alsoused for photodynamic therapy. Methylene blue is a phenothiazinecationic dye that is exemplified by its ability to specifically targetmitochondrial membrane potential. Rose-bengal and fluorescein arexanthene dyes that are well documented in the art for use inphotodynamic therapy. Rose bengal diacetate is an efficient,cell-permeant generator of singlet oxygen. It an iodinated xanthenederivative that has been chemically modified by the introductionof-acetate groups. These modifications inactivate both its fluorescenceand photosensitization properties, while increasing its ability to crosscell membranes. Once inside the cell, esterases remove the acetategroups and restore rose bengal to its native structure. Thisintracellular localization allows rose bengal diacetate to be a veryeffective photosensitizer.

In other aspects, the photosensitizers can be Diels-Alder adducts,dimethyl acetylene dicarboxylate adducts, anthracenediones,anthrapyrazoles, aminoanthraquinone, phenoxazine dyes,chalcogenapyrylium dyes such as cationic selena and tellurapyryliumderivatives, cationic imminium salts, and tetracyclines are othercompounds that also exhibit photoactive properties and can be usedadvantageously in photodynamic therapy. Other photosensitizers that donot fall in either of the aforementioned categories have other usesbesides photodynamic therapy, but are also photoactive. For example,anthracenediones, anthrapyrazoles, aminoanthraquinone compounds areoften used as anticancer therapies (i.e. mitoxantrone, doxorubicin).Chalcogenapyrylium dyes such as cationic selena- and tellurapyryliumderivatives have also been found to exhibit photoactive properties inthe range of about 600 to about 900 nm range, more preferably from about775 to about 850 nm. In addition, antibiotics such as tetracyclines andfluoroquinolone compounds have demonstrated photoactive properties.

Linkers/Enzyme Cleavage Site

Linkers of the invention are capable of linking two or more componentsof the photosensitizer composition together (e.g., a photosensitizer toanother photosensitizer, a photosensitizer to a binder, aphotosensitizer to a backbone, a binder to a backbone, a photosensitizerto a targeting moiety, or a binder to a targeting moiety). Any bondwhich is capable of linking the components such that they are stableunder physiological conditions for the time needed for administrationand treatment is suitable, but covalent linkages are advantageous. Thelink between two components may be direct, e.g., where a photosensitizeris linked directly to another photosensitizer, or indirect, e.g., wherea photosensitizer is linked to an intermediate, e.g., linked to abackbone, and that intermediate is linked to another photosensitizer. Alinker should function under conditions of temperature, pH, salt,solvent system, and other reactants that substantially retain thechemical stability of the photosensitizer, the backbone (if present),and the targeting moiety.

Linkers can comprise an enzyme cleavage site for a bacterial enzyme. Inone aspect of the invention, linker cleavage by a pathogen enzyme causesreduction of the quenching that results from the conformation adopted bythe multiple photosensitizers linked to one another. In another aspect,linker cleavage by a pathogen enzyme causes reduction of the quenchingthat results from inclusion of a binder (e.g., a quencher, see above).Without being bound by theory, target cells cause reduction of quenchingby the endogenous production of an enzyme which cleaves the linker. Thelinker enzyme cleavage site can be a beta-lactamase cleavage sitecomprising one or more beta lactam ring moieties. In a furtherembodiment, the linker comprises a penicillin or cephalosporin orfunctional derivative or fragment thereof.

One of the mechanisms utilized by bacteria to become resistant to anantibiotic involves the production of an enzyme that inactivates theantibiotic. An example of this type of resistance constitutes thebeta-lactamase enzymes, as described above. The beta-lactamase enzymescleave the four-membered (three carbon, one nitrogen) β-lactam(2-azetidinone) ring that constitutes the unique structural featureshared by the β-lactam antibiotics. This family of antibiotics includesthe penicillins, cephalosporins, penems, carbapenems, and monocyclicmonobactams, among others. Accordingly, in specific embodiments, thelinker comprises a penicillin, a cephalosporin, a carbapenem, or amonocyclic monobactam or a fragment thereof (e.g., comprising abeta-lactam ring). Such linkers can include derivatives of cephalosporinor other antibiotics, wherein such a derivative is cleaved bybeta-lactamase to the same or similar extent as the parent linker.Examples of derivatives of linkers include a linker as described herein,such as cephalosporin, which is conjugated to an additional moiety, suchas, for example, cephalosporin conjugated to aminothiophenol.

Another example of this type of resistance constitutes VanX, adipeptidase that cleaves D-Ala-D-Ala and catalyzes hydrolysis of theD-alanyl-D-alanine dipeptide normally used in wild-type peptidoglycanbiosynthesis. VanX-related proteins are involved in the production of avariant peptidoglycan (D-alanine-D-lactate) that results in resistanceof pathogenic bacteria to the antibiotic vancomycin. D-ala-D-alapeptidase has been found to be contained in the operon that encodesvancomycin resistance in Enterococcus faecalis and Enterococcus faecium.

Linkers can also be cross-linking agents that are homo- orhetero-bifunctional.

Many linkers react with an amine and a carboxylate, to form an amide, oran alcohol and a carboxylate to form an ester. Linkers are known in theart, see, e.g., M. Bodansky, “Principles of Peptide Synthesis”, 2nd ed.,referenced herein, and T. Greene and P. Wuts, “Protective Groups inOrganic Synthesis,” 2nd Ed, 1991, John Wiley, NY. Linkers should linkcomponent moieties stably, but such that there is only minimal or nodenaturation or deactivation of the photosensitizer or other linkedcomponent.

The photosensitizer compositions of the invention can be prepared bylinking the photosensitizers to one another or to other components usingmethods known in the art. A variety of linkers, including cross-linkingagents, can be used for covalent conjugation. Examples of cross-linkingagents include N,N′-dicyclohexylcarbodiimide (DCC),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),ortho-phenylenedimaleimide (o-PDM), and sulfosuccinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (Karpovskyet al. (1984) J. Exp. Med. 160:1686; Liu, M A et al. (1985) Proc. Natl.Acad. Sci. USA 82:8648). Other methods include those described by Paulusand Behring (1985) Ins. Mitt., 78:118-132; Brennan et al. (1985) Science229:81-83 and Glennie et al., (1987) J. Immunol, 139:2367-2375. A largenumber of linkers for peptides and proteins, along with buffers,solvents, and methods of use, are described in the Pierce Chemical Co.catalog, pages T-155 to T-200, 1994 (3747 N. Meridian Rd., RockfordIll., 61105, U.S.A.; Pierce Europe B.V., P.O. Box 1512, 3260 BA OudBeijerland, The Netherlands), the contents of which are herebyincorporated by reference.

DCC is a useful linker (Pierce #20320; Rockland, Ill.). DCC(N,N′-dicyclohexylcarbodiimide) is a carboxy-reactive cross-linkercommonly used as a linker in peptide synthesis. Another usefulcross-linking agent is SPDP (Pierce #21557), a heterobifunctionalcross-linker for use with primary amines and sulfhydryl groups. SPDPproduces cleavable cross-linking such that, upon further reaction, theagent is eliminated, so the photosensitizer can be linked directly to abackbone or molecular carrier. Other useful linking agents are SATA(Pierce #26102), for introduction of blocked SH groups for two-stepcross-linking (Pierce #26103), and sulfo-SMCC (Pierce #22322), reactivetowards amines and sulfhydryls. Other cross-linking and coupling agentsare also available from Pierce Chemical Co. (Rockford, Ill.).

Additional useful linking agents are hydrazines or hydrazinederivatives, compounds that are very soluble in water and soluble inalcohol. Hydrazines are corrosive and strong reducing agents, thoughthey constitute weaker bases than ammonia. Hydrazines are dibasic andform many salts, e.g., mono- and di-hydrochlorides, mono- anddi-nitrates, and two sulfates. The hydrazine resin has been found to bea novel and highly useful platform for polyamide synthesis. Thehydrazine resin is stable to elevated coupling temperatures, yet iscleaved rapidly at moderate temperatures by a wide range of nucleophilesfollowing a mild and selective oxidation protocol.

Additional compounds and processes, particularly those involving aSchiff base as an intermediate, for conjugation of proteins to otherproteins or to other compositions, for example, to reporter groups or tochelators for metal ion labeling of a protein, are disclosed in EP243,929 A2 (published Nov. 4, 1987).

Photosensitizers which contain carboxyl groups can be joined to lysines-amino groups in target polypeptides either by preformed reactiveesters (such as N-hydroxy succinimide ester) or esters conjugated insitu by a carbodiimide-mediated reaction. The same applies tophotosensitizers that contain sulfonic acid groups, which can betransformed to sulfonyl chlorides, which react with amino groups.Photosensitizers that have carboxyl groups can be joined to amino groupson the polypeptide by an in situ carbodiimide method or by hydrazine orhydrazine derivatives. Photosensitizers can also be attached to hydroxylgroups, of serine or threonine residues or to sulfhydryl groups, ofserine or threonine residues or to sulfhydryl groups of cysteineresidues.

Methods of joining components of a composition can useheterobifunctional cross linking reagents. These agents bind afunctional group in one chain and a different functional group in asecond chain. These functional groups typically are amino, carboxyl,sulfhydryl, and aldehyde. There are many permutations of appropriatemoieties that will react with these groups and with differentlyformulated structures, to join them together (described in the PierceCatalog and Merrifield et al. (1994) Ciba Found Symp. 186:5-20).

Generally, the photosensitizer compositions of the invention can beprepared by linking the photosensitizer to another photosensitizer, abinder, a targeting moiety, and/or a backbone using methods described inthe following Examples or by methods known in the art. A variety oflinkers can be used for covalent conjugation.

Yield from linking reactions can be assessed by spectroscopy of producteluting from a chromatographic fractionation in the final step ofpurification using known methods. The presence of unlinkedphotosensitizer and reaction products containing the photosensitizer canbe followed by the physical property that the photosensitizer absorbslight at a characteristic wavelength and extinction coefficient, soincorporation into products can be monitored by absorbance at thatwavelength or a similar wavelength. Linking of one or morephotosensitizer molecules to another photosensitizer, a binder, atargeting moiety or a backbone, shifts the peak of absorbance in theelution profile in fractions eluted using sizing gel chromatography,e.g., with the appropriate choice of Sephadex G50, G100, or G200 orother such matrices (Pharmacia-Biotech, Piscataway N.J.). Choice ofappropriate sizing gel, for example Sephadex gel, can be determined bythat gel in which the photosensitizer elutes in a fraction beyond theexcluded volume of material too large to interact with the bead, i.e.,the uncoupled starting photosensitizer composition interacts to someextent with the fractionation bead and is concomitantly retarded to someextent.

Determining which gel to use can be predicted from the molecular weightof the uncoupled photosensitizer. The successful reaction products ofphotosensitizer compositions coupled to additional moieties generallyhave characteristic higher molecular weights, causing them to interactwith the chromatographic bead to a lesser extent, and thus appear infractions eluting earlier than fractions containing the uncoupledphotosensitizer substrate. Unreacted photosensitizer substrate generallyappears in fractions characteristic of the starting material, and theyield from each reaction can thus be assessed both from size of the peakof larger molecular weight material, and the decrease in the peak ofcharacteristic starting material. The area under the peak of the productfractions is converted to the size of the yield using the molarextinction coefficient.

The product can be analyzed using NMR, integrating areas of appropriateproduct peaks, to determine relative yields with different linkers. Ared shift in absorption of a photosensitizer of several nm has oftenbeen observed following coupling to a polyamino acid. Linking to alarger moiety such as a protein might produces a comparable shift, aslinking to an antibody resulted in a shift of about 3-5 nm in thatdirection compared to absorption of the free photosensitizer. Relevantabsorption maxima and extinction coefficients in 0.1M NaOH/1% SDS are,for chlorin e6, 400 nm and 150,000 M⁻¹ cm⁻¹, and for benzoporphyrinderivative, 430 nm and 61,000 M⁻¹, cm⁻¹.

The linker (L) can comprise at least one beta-lactam ring moiety or afragment or derivative thereof which forms a beta-lactamase cleavablesite.

In particular aspects, the linker (L) comprises a beta-lactamantibiotic, or functional derivative or fragment thereof which iscleavable by a beta-lactamase enzyme or fragment thereof. Thebeta-lactam antibiotic (or functional derivative or fragment thereof)can be any suitable beta-lactam antibiotic which is obtained from anynatural, commercial or synthetic source.

In one aspect, the linker (L) comprises a penicillin or fragment thereofhaving the general known core nucleus structure:

and which is selected from: a narrow spectrum penicillin, such as,benzthine penicillin, benzylpenicillin (penicillin G),phenoxymethylpenicillin (penicillin V), procaine penicillin, andoxacillin; a narrow spectrum penicillinase-resistant penicillin, suchas, methicillin, dicloxacillin, and flucloxacillin; a narrow spectrumbeta-lactamase-resistant penicillin, such as, temocillin; a moderatespectrum penicillin, such as, amoxicillin and ampicillin; a broadspectrum penicillin, such as, co-amoxiclav; and an extended spectrumpenicillin, such as, carboxypenicillins, ureidopenicillins, azlocillin,carbenicillin, ticarcillin, mezlocillin and piperacillin, or anyfragment or derivative of a penicillin, and wherein R corresponds to thedifferent moieties of the above indicated penicillins.

In another aspect, the linker (L) comprises a cephalosporin having thegeneral known core nucleus structure:

and which is selected from: a first generation cephalosporin, such as,Cefacetrile (cephacetrile), Cefadroxil (cefadroxyl; Duricef®), Cefalexin(cephalexin; Keflex®), Cephaloglycin, Cefalonium (cephalonium),Cefaloridine (cephaloradine), Cefalotin (cephalothin; Keflin®),Cefapirin (cephapirin; Cefadryl®), Cefatrizine, Cefazaflur, Cefazedone,Cefazolin (cephazolin; Ancef®, Kefzol®), Cefradine (cephradine;Velosef®), Cefroxadine, Ceftezole; a second generation cephalosporin,such as, Cefaclor (e.g., Ceclor®, Distaclor®, Keflor®, Raniclor®),Cefonicid (e.g, Monocid®), Cefprozil (e.g., cefproxil; Cefzil®),Cefuroxime (e.g., Zinnat®, Zinacef®, Ceftin®, Biofuroksym®), Cefuzonam,Cefmetazole, Cefotetan, Cefoxitin, Carbacephems (e.g., loracarbef(Lorabid®)), Cephamycins (e.g., cefbuperazone, cefmetazole (Zefazone®),cefminox, cefotetan (Cefotan®), cefoxitin (Mefoxin®)); a secondgeneration cephamycin, such as, cefotetan or cefoxitin; a thirdgeneration cephalosporin, such as, Cefcapene, Cefdaloxime, Cefdinir(Omnicef®), Cefditoren, Cefetamet, Cefixime (Suprax®), Cefmenoxime,Cefodizime, Cefotaxime (Claforan®), Cefpimizole, Cefpodoxime (Vantin®,PECEF), Cefteram, Ceftibuten (Cedax), Ceftiofur, Ceftiolene, Ceftizoxime(Cefizax®), Ceftriaxone (Rocephin®), Cefoperazone (Cefobid), Ceftazidime(Fortum®, Fortaz®), or Oxacephems (e.g. latamoxef); or a fourthgeneration cephalosporin, such as, cefepime (Maxipime®), cefclidine,cefluprenam, cefoselis, cefozopran, cefpirome, or cefquinome, orcefpirome, or any fragment or derivative of a cephalosporin, wherein theR and X correspond to the different moieties of the above indicatedcephalosporins.

In another aspect, the linker (L) can also be a carbapenem, such as,imipenem, meropenem, ertapenem, faropenem, or doripenem, or any fragmentor derivative of a carbapenem.

In a particular aspect, the linker (L) of the invention comprises acephalosporin, or fragment or derivative thereof. The photosensitizercompositions of the invention can comprise a cephalosporin linker (orfragment and/or derivative thereof) coupling two photosensitizers inaccordance with the following structure:

wherein X can be any known cephalosporin substituent or derivativethereof, and wherein EtNBS is benzophenothiazinium chloride.

In another particular embodiment, the present invention provides aphotosensitizer composition comprising a cephalosporin linker (orfragment and/or derivative thereof) coupling two benzophenothiaziniumchloride (EtBNBS) photosensitizers in accordance with the followingstructure:

The cephalosporin linker may be any cephalosporin known in the art, suchas those listed above, or any functional derivative or fragment thereof.One of ordinary skill in the art will appreciate that there are numerousknown methods and chemical approaches for preparing and obtainingfragments and derivatives of cephalosporins, some of which are disclosedin U.S. Pat. No. 6,599,893 (to Glinka), U.S. Pat. No. 6,093,712 (toMatiskella), U.S. Pat. No. 5,827,845 (to Shiokawa et al.) and U.S. Pat.No. 6,329,363 (to Dahnke), each of which are incorporated in theirentireties herein by reference.

Specific combinations of linker and photosensitizer contemplatedaccording to the invention include, but are not limited to cephalosporinwith two EtNBS photosensitizers; cephalosporin with EtBNS and a blackhole quencher (e.g., such as BHQ-3); cephalosporin with Cy3 and Cy5;cephalosporin with Cy5 and a black hole quencher; and cephalosporin withOregon Green and Texas Red. These specific combination ofphotosensitizer and linker can be readily made by one of skill in theart according to the methods described herein.

Binders

The photosensitizer compositions of the invention can also include oneor more binders. The binder, without limitation, may be a peptide, acyclic peptide, a polypeptide, a peptidomimetic, a protein, a fusionprotein, a hybrid molecule, another photosensitizer or a dimer,multimer, or a conjugate of the above that binds or quenches, and, thus,may inhibit, suppress, neutralize, or decrease activity of, thephotosensitizer. The binder may also include, without limitation, anaturally occurring inhibitor, a receptor, a soluble receptor, anantibody, a polyclonal antibody, a monoclonal antibody, a bispecificantibody, an antibody fragment, a single chain antibody, anti-idiotypeantibodies, a peptabody, a peptide, an oligopeptides, anoligonucleotide, a cyclic peptide (i.e., a peptide that is circular innature), a peptide-lipid conjugate, a hormone, an antigen, an epitope, areceptor, a chemokine, a nucleic acid, a ligand or a dimer, multimer, ora conjugate of the above. Naturally occurring binders are binders thatquench the photosensitizer and are found in nature.

In one aspect, the binder comprises a fluorophore. The property thatrenders a fluorophore (or any other binder) a suitable quencher is thecapability of absorbing energy from the activated or activatablephotosensitizer.

Fluorophores of the present invention can be any known in the art,including photosensitizers, fluorescent dyes, and photoactive dyes.

Photosensitizer binders can be any known in the art, as previouslydescribed. For example, hematoporphyrin derivatives have been used asfluorescent probes to investigate the development of humanatherosclerotic plaques (Spokojny (1986) J. Am. Coll. Cardiol.8:1387-1392). A photosensitizer acting as a binder can have a differentexcitation wavelength than a photosensitizer acting to produce acytotoxic effect on a pathogen or host cell infected with a pathogen.

Fluorescent dyes, which can be used as photosensitizers or binders ofthe invention, can be any known in the art, including, but not limitedto 6-carboxy-4′,5′-dichloro-2′, 7′-dimethoxyfluorescein succinimidylester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein;6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein;5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether,-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluoresceinsuccinimidyl ester; 6-carboxyfluorescein succinimidyl ester;5-(and-6)-carboxyfluorescein succinimidyl ester;5-(4,6-dichlorotriazinyl) aminofluorescein; 2′,7′-difluorofluorescein;eosin-5-isothiocyanate; erythrosin-5-isothiocyanate;6-(fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester;6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid or succinimidylester; fluorescein-5-EX succinimidyl ester;fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green®500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® 514 carboxylic acid;Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreen™ carboxylic acid, succinimidyl ester or hydrochloride; RhodamineGreen™ carboxylic acid, trifluoroacetamide or succinimidyl ester;RhodamineGreen™-X succinimidyl ester or hydrochloride; Rhodol Green™carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester;bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl ester);5-(and-6)-carboxynaphthofluorescein, 5-(and-6)-carboxynaphthofluoresceinsuccinimidyl ester; 5-carboxyrhodamine 6G hydrochloride;6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidylester; 6-carboxyrhodamine 6G succinimidyl ester;5-(and-6)-carboxyrhodamine 6G succinimidyl ester;5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein succinimidyl ester orbis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine;6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine;5-carboxytetramethylrhodamine succinimidyl ester;6-carboxytetramethylrhodamine succinimidyl ester;5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;6-carboxy-X-rhodamine succinimidyl ester; 5-(and-6)-carboxy-X-rhodaminesuccinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt;Lissamine™ rhodamine B sulfonyl chloride; malachite greenisothiocyanate; NANOGOLD® mono(sulfosuccinimidyl ester); QSY® 21carboxylic acid or succinimidyl ester; QSY® 7 carboxylic acid orsuccinimidyl ester; Rhodamine Red™-X succinimidyl ester;6-(tetramethylrhodamine-5- (and-6)-carboxamido)hexanoic acidsuccinimidyl ester; tetramethylrhodamine-5-isothiocyanate;tetramethylrhodamine-6-isothiocyanate;tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl;Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt;Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; orX-rhodamine-5-(and-6)-isothiocyanate.

Fluorescent dyes can also include, for example, bodipy dyes commerciallyavailable from Molecular Probes, including, but not limited to BODIPY®FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630/650-X STPester; BODIPY® 650/665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidylester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acidsuccinimidyl ester;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsulfosuccinimidyl ester or sodium salt;6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid;6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid or succinimidyl ester;N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteicacid, succinimidyl ester or triethylammonium salt;6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid;4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester;4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionicacid;4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionicacid succinimidyl ester;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diazas-indacene-3-yl)phenoxy)acetyl) amino)hexanoic acid or succinimidylester; and6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester.

Fluorescent dyes of the present invention can also be, for example,alexa fluor dyes commercially available from Molecular Probes, includingbut not limited to Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430carboxylic acid; Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647carboxylic acid; Alexa Fluor® 660 carboxylic acid; or Alexa Fluor® 680carboxylic acid.

Fluorescent dyes of the present invention can also be, for example, cydyes commercially available from Amersham-Pharmacia Biotech, including,but not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; andCy 7 NHS ester.

Photoactive dyes, which can also be used as binders or photosensitizersof the invention, can be any photosensitizer known in the art which willfluoresce but will not necessarily produce a reactive species inphototoxic amounts when illuminated. Depending on the wavelength andpower of light administered, a photosensitizer can be activated tofluoresce and, therefore, act as a photoactive dye, but not produce aphototoxic effect unless, in some cases, the wavelength and power oflight is suitably adapted to induce a phototoxic effect.

Throughout this specification, any reference to a binder should beconstrued to refer to each of the binders identified and contemplatedherein and to each biologically equivalent molecule. “Biologicallyequivalent” means compositions of the present invention which arecapable of preventing action of the photosensitizer in a similarfashion, but not necessarily to the same degree.

Targeting Moieties

The inventive photosensitizer compositions of the invention alsocontemplate being coupled to one or more targeting moieties, whichincrease the specificity of the photosensitizer composition for itstarget, and can be used for a variety of purposes, including, forexample, targeting the photosensitizer composition of the invention toreach a closer proximity to the target cells or enzymes (e.g.,beta-lactamase) which are of interest to be assayed or measured.Targeting moieties include antibody and antibody fragments, peptides,and hormones. In one aspect of the invention, the targeting moiety canbe a polypeptide (e.g., a human polypeptide such as poly-lysine or serumalbumin). Alternatively, the targeting moiety can be a smallanti-microbial peptide (SAMPs) (i.e., a peptide containing less than 60amino acid residues), such as, for example, histatins, defensins,cecropins, magainins, Gram positive bacteriocins, and peptideantibiotics. Many SAMP's are in the range of 20-40 amino acid residuesin length. SAMP's are naturally occurring peptides, and are made by awide variety of organisms. Many SAMP's have a broad spectrum ofantimicrobial activity, and, e.g., can kill more than one species, andin some cases can kill distantly related species, e.g. Gram negative andGram positive bacterial species.

The targeting moiety can bind to a defined population of cells. Invarious aspects, it can bind a receptor, an oligonucleotide, anenzymatic substrate, an antigenic determinant, or other binding sitepresent on or in the target cell population. Accordingly, the targetingmoiety can be a molecule or a macromolecular structure that targetsspecific cells, for example, macrophages, or that interacts with apathogen. Some photosensitizers target macrophages directly (see, e.g.,Korbelik et al., Cancer Res. 51:2251-2255, 1991).

Moieties, either alone or when incorporated into a conjugate, as in asupramolecular structure (e.g., a liposome, a micelle, a lipid vesicle,or the like), can be used to specifically target macrophages by certainreceptors. Thus, a ligand for such receptors can be used as a targetingmoiety. For example, the following receptors can be used to targetmacrophages: the complement receptor (Rieu et al., J. Cell Biol.127:2081-2091, 1994), the scavenger receptor (Brasseur et al.,Photochem. Photobiol. 69:345-352, 1999; Suzuki et al., Nature386:292-296, 1997; Sarkar et al., Mol. Cell. Biochem. 156:109-116,1996), the transferrin receptor (Dreier et al., Bioconjug. Chem.9:482-489, 1998; Hamblin et al., J. Photochem. Photobiol. 26:4556, 1994;Clemens et al., J. Exp. Med. 184:1349-1355, 1996), the Fc receptor(Rojanasakul et al., Pharm. Res. 11:1731-1733, 1994; Harrison et al.,Pharm Res. 11:1110-4, 1994). The mannose receptor is particularlyimportant for macrophage recognition of foreign material and has beenused successfully to target molecules to macrophages (Frankel et al.,Carbohydr. Res. 300:251-258, 1997; Chakrabarty et al., J. Protozool.37:358-364, 1990; Mistry et al., Lancet 348:1555-1559, 1996; Liang etal., Biochim. Biopys. Acta 1279:227-234, 1996; Sarkar et al., Mol. CellBiochem. 156:109-116, 1996). Toll or toll-like receptors are alsopresent on macrophages and are useful targets (Brightbill et al.,Science 285:732-736, 1999).

The photosensitizer compositions of the invention can comprise targetingmoieties which target the former to macrophages. Such targeting moietiescan include low density lipoproteins (Mankertz et al., Biochem. Biophys.Res. Commun. 240:112-115, 1997; von Baeyer et al., Int. J. Clin.Pharmacol. Ther. Toxicol. 31:382-386, 1993), very low densitylipoproteins (Tabas et al., J. Cell Biol. 115:1547-1560, 1991), mannoseresidues (as mentioned above) and other carbohydrate moieties (Pittet etal., Nucl. Med. Biol. 22:355-365, 1995), poly-cationic molecules (e.g.,poly-Llysine; Hamblin et al., J. Photochem. Photobiol. 26:45-56, 1994),emulsions (Khopade et al., Pharmazie 51:558-562, 1996), aggregatedalbumin (Hamblin et al., J. Photochem. Photobiol. 26:45-56, 1994),biodegradable microspheres (Oettinger et al., J. Interferon CytokineRes. 19:33-40, 1999), non-biodegradable microspheres (Schroder, MethodsEnzymol 112:116-128, 1985), nanoparticles (Lobenberg et al., AIDS Res.Hum. Retroviruses 12:1709-1715, 1996); Venier-Julienne et al., J. DrugTarget. 3:23-29, 1995; Schafer et al., J. Microencapsul. 11:261-269,1994; Gaspar et al., Ann. Trop. Med. Parasitol 86:41-49, 1992),liposomes (Bakker-Woudenberg et al. J. Drug Target. 2:363-371, 1994;Meyers et al., Exp. Lung Res. 19:1-19, 1993; Betageri et al., J. Pharm.Pharmacol. 45:48-53, 1993; Muller et al., Biochim. Biophys. Acta.986:97-105, 1989; Kole et al., J. Infect. Dis. 180:811-820, 1999),macrophage-specific cytokines (Biragyn et al., Nat. Biotechnol.17:253-258, 1999; Chan et al., Blood 86:2732-2740, 1995), erythrocytes(Magnani et al., J. Leukoc. Biol. 185:717-730, 1997), antibodiesrecognizing components of the tuberculous phagosome like Nrampl(Gruenheid et al., J. Exp. Med. 185:717-730, 1997), a 2-macroglobulin(Chu et al., J. Immunol. 152:1538-1545, 1994).

A targeting moiety can be directed to a pathogen. In addition, certainstructural features of enzymes can be targeted, such as the hydrophobicpocket of the Mycobacterium tuberculosis enzyme inhA (Dessen, et al.(1995) Science 267:1638-1641). Alternatively, host molecules that targetthe bacteria, such as anti-microbial peptides (e.g., granulysin), can beused in photosensitizer compositions of the invention (Stenger et al.,Science 282:121-125, 1998).

A targeting moiety can be used alone or in combination, particularly totarget both macrophages and the intracellular pathogen. Manipulations ofthe host cell can also complement the photosensitizer (Collins et al.,J. Cell Sci. 110:191-200, 1997; Korbelik et al., Br. J. Cancer75:202-207, 1997; Krosl et al., Cancer Res. 56:3281-3286, 1996).

The targeting moiety can be a polypeptide. The polypeptide may belinear, branched, or cyclic. The targeting moiety can include apolypeptide having an affinity for a polysaccharide target, for example,a lectin (such as a seed, bean, root, bark, seaweed, fungal, bacterial,or invertebrate lectin). Particularly useful lectins includeconcanavalin A, which is obtained from jack beans, and lectins obtainedfrom the lentil, Lens culinaris.

Desirable characteristics for the targeting moieties include:specificity for one or more unwanted target organisms or componentsthereof (e.g. cell surface receptors), affinity and avidity for suchorganisms, and stability with respect to conditions of couplingreactions and the physiology of the organ or tissue of use. Specificityneed not be narrowly defined, e.g., it may be desirable for a targetingmolecule to have affinity for a broad range of target organisms, such asall Gram negative bacteria. The targeting moiety, when incorporated intoa composition of the invention, should be nontoxic to the cells of thesubject.

Targeting moieties can be selected from the sequences of naturallyoccurring proteins and peptides, from variants of these peptides, andfrom biologically or chemically synthesized peptides. Naturallyoccurring peptides which have affinity for one or more target organismcan provide sequences from which additional peptides with desiredproperties, e.g., increased affinity or specificity, can be synthesizedindividually or as members of a library of related peptides. Suchpeptides can be selected on the basis of affinity for the targetorganism.

Naturally occurring peptides with affinity for target organisms usefulin methods and compounds of the invention include, aptomers, salivaryproteins, e.g., histatins, microbially-elaborated proteins, e.g.,bacteriocins, peptides that bind and/or kill species that are closelyrelated to the producing strains; and proteins produced by animalspecies, such as defensins, which are produced by mammals, and thececropins and magainins, produced by moths and amphibia, respectively.

As mentioned briefly above, histatins, defensins, cecropins andmagainins are examples of a class of polypeptides found widely innature, which share the characteristics of small size (generallyapproximately 30 amino acid residues, and between 10 residues and 50residues), broad specificity of anti-microbial activity, and lowaffinity for target organisms.

Histatins are a family of histidine-rich cationic polypeptides whichhave bactericidal and candidacidal properties and are constituents ofnormal human saliva (Oppenheim, G. G. et al., J. Biol. chem.263:7472-747, 1988). Their mechanism of action is thought to involve acombination of alpha-helical conformation and cationic charge leadingthem to insert between the polar head groups in the bacterial cell wall(Raj, P. A. et al., J. Biol. Chem. 269:9610-9619, 1994).

Bacteriocins, which are proteins produced by bacteria and which killother strains and species of bacteria (Jack, R. W. et al., Microbiol.Rev. 59:171-200, 1995) can be used as targeting moieties. An exemplaryGram positive bacteriocin is nisin, produced by Lactococcus lactis andaccorded GRAS status (generally regarded as safe) by the Food and DrugAdministration for application to food preservation.

The bacteriocins, nisin, subtilin, epidermin, gallidermin, salivarin,and lacticin exemplify the “lantibiotic” class of Gram positivebacteriocins, which is defined as those bacteriocins in which one ormore cysteine residues are linked to a dehydrated serine or threonine toform a thioether-linked residue known as lanthionine (Lan) orthreo-β-methyllanthionine (MeLan). These are post-translationalmodifications found in these anti-microbial peptides by the producingcell. Lantibiotics contain leader peptide sequences of 18-24 residues,which are cleaved to yield an active antimicrobial peptide of about22-35 residues. Growth of the producing bacterial species, andpreparation and purification of bacteriocins are performed by publishedprocedures and techniques which can be carried out by one of skill inthe art. For example, Yang, R. et al., Appl. and Env. Microbiol 58:3355-3359, 1992, describe purification of bacteriocins from each of 4genera of lactic acid bacteria, by optimizing absorption onto theproducing cells, followed by use of low pH for selective elution ofgreatly enriched bacteriocin fractions. Mutant forms of each of thebacteriocins, nisin, produced by Lactococcus lactis, and subtilin,produced by Bacillus subtilis have more desirable properties than theparental wild-type forms (Liu, W. and N. Hansen, J. Biol. Chem.267:25,078-25,085, 1992). Procedures for isolation of appropriate genesand for mutagenesis and selection of strains carrying desirablemutations are found in Maniatis, T. et al, 1982, Molecular Cloning: aLaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,and in the subsequent second edition, Sambrook, J. et al., 1989.

Anti-microbial peptides are produced by a variety of animals (Saberwal,G. and R. Nagaraj, Biochim. Biophys. Act. 1197:109-131, 1994). Anexample is a peptide of the cecropin family produced by Cecropia moths.Several cecropins contain 37 residues, of which 6 are lysine. Cecropinsare active against both Gram positive and Gram negative bacteria. Otherinsect-produced peptides include apidaecin (from honeybees), andropin(from fruit flies), and cecropin family members from bumble bees, fruitflies, and other insects.

The defensins are produced by mammals, including humans, and aregenerally about 29-34 residues in length, and the magainins (about 23residues) are produced by amphibia such as Xenopus laevis. Defensinsfrom human (HNP-1,-2,-3 and 4), guinea pig (GPNP), rabbit (NP-1, -2,-3A, -3B, -4 and -5) and rat (NP-1, -2, -3 and -4) share a significantnumber of regions of homology. Defensins can have antimicrobial activityagainst Gram positive bacteria or Gram negative bacteria and fungi, withminimal inhibitory concentrations in the mM range. Rabbit NP-1 and NP-2are more potent antibacterial agents than others in this family. Othermammalian anti-microbial peptides include murine cryptdin, bovinegranulocyte bactenecin and indolicidin, and seminal-plasmin from bovinesemen. Additional amphibial anti-microbials include PGLA, XPF, LPF, CPG,PGQ, bombinin from Bombina variegata, the bombinin-like peptides BLP-1,-2, -3 and -4 from B. orientalis, and brevinins from Rana esculenta.Invertebrates such as the horseshoe crab can be a source ofanti-microbial peptides such as the tachyplesins (I, H and III) and thepolyphemusins (I and II).

Peptides in these families of antimicrobial agents are generallycationic, and can have a broad antimicrobial spectrum, including bothantibacterial and antifungal activities. The addition of positivelycharged residues can enhance antimicrobial specific activity severalfold. The positive charges are thought to assist in the insertion of thepeptides into the membranes of the susceptible organisms, in whichcontext the peptide molecules can form pores and cause efflux of ionsand other metabolites. Structural studies of the Moses sole fishneurotoxin 33 residue peptide pardaxin, for example, reveals thatsuccinylated pardaxin inserts into erythrocyte and model membranes moreslowly than unmodified pardaxin. (Shai, Y et al., J. Biol. Chem. 265:20, 202-20, 209, 1990). The positively charged magainin molecule candisrupt both the metabolism of E. coli and the electric potential of themitochondrion (Westerhoff, H. V., et al., Proc. Natl. Acad. Sci.86:6597-6601, 1989).

Novel peptides, for example, a cecropin-melittin hybrid, and syntheticDenantiomers, have antimicrobial activity (Merrifield, R. B. et al.,“Antimicrobial peptides,” Ciba Foundation Symp. 186, John Wiley,Chichester, pp. 5-26, 1994). One such synthetic cecropin-melittinpeptide is 5-fold more active against Mycobacterium smegmatis thanrifampin.

Targeting moieties can be plant proteins with affinities for particulartarget organisms, for example, a member of the lectin protein familywith affinity for polysaccharides. Targeting moieties can be syntheticpeptides, such as polylysine, polyarginine, polyornithine, and syntheticheteropolypeptides that comprise substantial proportions of suchpositively charged amino acid residues. Such peptides can be chemicallysynthesized or produced biologically in recombinant organisms, in whichcase the targeting moiety peptide can be produced as part of a largerprotein, for example, as the N-terminus residues, and cleaved from thatlarger protein. Polypeptides suitable as “backbone” moieties are alsosuitable as target moieties, if they have sufficient affinity for thetarget organism. Considerations described are thus appropriate to thegeneral consideration of a targeting moieties.

Targeting moieties need not be limited to peptide compositions, but canbe lectins, polysaccharides, steroids, and metalloorganic compositions.Targeting moieties can be comprised of compositions that are composedboth of amino acids and sugars, such as mucopolysaccharides. A usefultargeting moiety can be partially lipid and partially peptide in nature,such as low density lipoprotein. Serum lipoproteins especially highdensity and low density lipoproteins (HDL and LDL) can bind to bacterialsurface proteins (Emancipator, K. et al., Infect. Immun. 60:596-601,1992). HDL, and especially reconstituted HDL, neutralizes bacteriallipopolysaccharide both in vitro and in vivo (Wurfel M M et al., J. Exp.Med. 181:1743-1754, 1995). Endogenous LDL can protect against the lethaleffects of endotoxin and Gram negative infection (Netea, M., et al., J.Clin. Invest. 97:1366-1372, 1996). The appropriate binding features ofthe lipoproteins to bacterial surface components can be identified bymethods of molecular biology known in the art, and the binding featureof lipoproteins can be used as the targeting moiety in photosensitizercompositions of the present invention.

Molecules, e.g., peptides, other than antibodies and members of a highaffinity ligand pairs, can be used to target a photosensitizercomposition according to the invention to a target organism. Targetingmoieties can be modified or refined. Once an example of a targetingmoiety of reasonable affinity has been provided, one skilled in the artcan alter the disclosed structure (of a polylysine polypeptide, forexample), by producing fragments or analogs, and testing the newlyproduced structures for modification of affinity or specificity.Examples of methods which allow the production and testing of fragmentsand analogs are discussed in U.S. Pat. No. 6,462,070.

In aspects pertaining to diagnostic methods, the skilled artisan willappreciate that the targeting moieties described herein can beconfigured such that they do not interfere with the activation ordequenching of the photosensitizers associated with linker cleavage.

Backbones

Photosensitizer compositions according to the invention include those inwhich a “backbone” moiety, such as a polyamino acid, is linked to aphotosensitizer and/or to a binder and/or to a targeting moiety.Additionally, the backbone can itself be a targeting moiety, e.g.polylysine.

Inclusion of a backbone in a composition with a photosensitizer and/orbinder and/or targeting moiety can provide a number of advantages,including the provision of greater stoichiometric ranges ofphotosensitizers and/or binders and/or targeting moieties coupled perbackbone. If the backbone possesses intrinsic affinity for a targetorganism, the affinity of the composition can be enhanced by coupling tothe backbone. Furthermore, the specific range of organisms that can betargeted with one composition can be expanded by coupling two or moredifferent targeting moieties to a single photosensitizer-backbonecomposition. However, it will be appreciated that in embodimentspertaining to diagnostic methods, the backbone should be configured suchthat it does not interfere with the activation or dequenching of thephotosensitizers following linker cleavage.

Peptides useful in the methods and compounds of the invention for designand characterization of backbone moieties include poly-amino acids whichcan be homo- and hetero-polymers of L-, D-, racemic DL- or mixed L- andD-amino acid composition, and which can be of defined or random mixedcomposition and sequence. Examples of naturally-occurring peptides withmixed D and L amino acid residues include bacitracin and tyrocidin.These peptides may be modeled after particular natural peptides, andoptimized by the technique of phage display and selection for enhancedbinding to a chosen target, so that the selected peptide of highestaffinity is characterized and then produced synthetically.

Further modifications of functional groups can be introduced forpurposes, for example, of increased solubility, decreased aggregation,and altered extent of hydrophobicity. Examples of non-peptide backbonesinclude nucleic acids and derivatives of nucleic acids, such as: DNA,RNA and peptide nucleic acids; polysaccharides and derivatives such asstarch, pectin, chitins, celluloses and hemi-methylated celluloses;lipids such as triglyceride derivatives and cerebrosides; syntheticpolymers such as polyethylene glycols (PEGs) and PEG star polymers;dextran derivatives, polyvinyl alcohols,N-(2-hydroxypropyl)-methacrylamide copolymers, poly (DL-glycolicacid-lactic acid); and compositions containing elements of any of theseclasses of compounds.

Administration of the Photosensitizer Compositions of the Invention

In certain aspects, the photosenstizer compositions of the invention canbe used therapeutically, i.e., for photodynamic therapy of bacterialinfections. The photosensitizer compositions of the invention can bedelivered to a subject in a free form, i.e., in solution. Alternativelythe compositions can be delivered in various formulations including, butnot limited to, liposome, peptide-bound, polymer-bound, ordetergent-containing formulations. Those of ordinary skill in the artare well able to generate and administer such formulations. Thecomposition should be soluble under physiological conditions, in aqueoussolvents containing appropriate carriers or excipients, or in othersystems, such as liposomes, that may be used to administer the conjugateto a subject.

Photosensitizer compositions that are somewhat insoluble in an aqueoussolvent can be applied in a liposome, or a time release fashion, suchthat illumination can be applied intermittently using a regimen ofperiods of illumination alternating with periods of non-illumination.Other regimens contemplated are continuous periods of lower levelillumination, for which a time-release formulation is suitable.

A composition of the present invention can be administered in atherapeutically effective amount by a variety of methods known in theart, including orally and topically. In one aspect, a photosensitizercomposition of the invention may be administered parenterally. Thephrase “administered parenterally” as used herein means modes ofadministration other than oral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andinfrasternal injection and infusion. As used herein, a “therapeuticallyeffective amount” refers to that amount of a photosensitizer compositionthat, when administered to a subject, is sufficient to decrease theactivity of a pathogen such that an infection is reduced or alleviated.

As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. Aphotosensitizer composition according to the invention can be containedin a pharmaceutically acceptable excipient or carrier. Included, withoutlimitation, are any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Advantageously, the carrier is suitable for oral,intravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound may be coated in amaterial to protect the compound from the action of acids and othernatural conditions that may inactivate the compound.

In one aspect, the carrier may protect the compound against rapidrelease, for example, a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In another aspect of the invention, the photosensitizer compositions canbe administered by combination therapy, i.e., combined with otheragents. For example, the combination therapy can include a compositionof the present invention with at least one other photosensitizer, atleast one antibiotic, or other conventional therapy.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

One of ordinary skill in the art can determine and prescribe theeffective amount of the pharmaceutical composition as needed. Forexample, one could start doses of the known or novel photosensitizercomposition levels lower than that indicated in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. For example, the dosage may range from 0.1mg/kg to 10 mg/kg depending on the therapeutic agent used.

The iterations delineated above are not intended as limiting withrespect to the nature of the conjugate photosensitizer compositions ofthe invention, or to a particular route of the administration.

Photoactivation/Illumination

The photosensitizer compositions of the invention are photoactivated inboth therapeutic and diagnostic uses according to the invention. Fortherapeutic uses, administration of a photosensitizer compositionaccording to the invention is typically followed by a sufficient periodof time to allow accumulation thereof at the target site. Uponencountering the target pathogen and/or infected host cell to be treatedor evaluated diagnostically, the enzyme cleavage site of the linker iscleaved by enzymes produced by the pathogen. Of note, the enzymes can besecreted by, can be internal to, or can reside on the surface, or withinthe cell wall space of a pathogen. Once the linker is cleaved, thephotosensitizers are rendered in an unquenched state. Thephotosensitizers can, subsequently, be activated by irradiation. This isaccomplished by applying light of a suitable wavelength and intensity,for an effective length of time, at the site of the infection fortherapeutic uses, or at the site of reaction for diagnostic uses, e.g.,reaction vessel. As used herein, “irradiation” refers to the use oflight to induced a chemical reaction of a photosensitizer.

Photoactivating dosages depend on various factors, including the amountof the photosensitizer administered, the wavelength of thephotoactivating light, the intensity of the photoactivating light, andthe duration of illumination by the photoactivating light. Thus, thedose can be adjusted to a therapeutically effective dose or to a dosesuitable for diagnostics by adjusting one or more of these factors. Suchadjustments are within the level of ordinary skill in the art.

Irradiation of the appropriate wavelength for a given compound may beadministered by a variety of methods. Methods for irradiation include,but are not limited to, the administration of laser, nonlaser, or broadband light. Irradiation can be produced by extracorporeal orintraarticular generation of light of the appropriate wavelength. Lightused in the invention may be administered using any device capable ofdelivering the requisite power of light including, but not limited to,fiber optic instruments, arthroscopic instruments, or instruments thatprovide transillumination. With therapeutic embodiments, delivery of thelight to a recessed, or otherwise inaccessible physiological locationcan be facilitated by flexible fiber optics (implicit in this statementis the idea that one can irradiate either a broad field, such as thelung or a lobe of the lung, or a narrow field where bacterial cells mayhave localized).

The photosensitizer compositions of the invention, in some aspects, canbe stable during the course of at least a single round of treatment ordiagnostic use (e.g., detection of and/or quantitation of beta-lactamaseactivity) by continued or pulsed irradiation, during which thephotosensitizer within the composition would, advantageously, berepeatedly excited to the energized state, undergoing multiple rounds ofgeneration of singlet oxygen.

The suitable wavelength, or range of wavelengths, will depend on theparticular photosensitizer(s) used, and can range from about 350 nm toabout 550 nm, from about 550 nm to about 650 nm, from about 650 nm toabout 750 nm, from about 750 nm to about 850 nm and from about 850 nm toabout 950 nm.

In some aspects, target tissues are illuminated with red light. Giventhat red and/or near infrared light best penetrates mammalian tissues,photosensitizers with strong absorbances in the range of about 600 nm toabout 900 nm can be suitable for activation of administeredphotosensitizers of the invention. For photoactivation, the wavelengthof light is matched to the electronic absorption spectrum of thephotosensitizer so that the photosensitizer absorbs photons and thedesired photochemistry can occur. Wavelength specificity forphotoactivation generally depends on the molecular structure of thephotosensitizer. Photoactivation can also occur with sub-ablative lightdoses. Determination of suitable wavelength, light intensity, andduration of illumination is within ordinary skill in the art.

With therapeutic uses, the effective penetration depth, δ_(eff), of agiven wavelength of light is a function of the optical properties of thetissue, such as absorption and scatter. The fluence (light dose) in atissue is related to the depth, d, as: e^(−d)/δ_(eff). Typically, theeffective penetration depth is about 2 to 3 mm at 630 nm and increasesto about 5 to 6 nm at longer wavelengths (about 700 to about 800 nm)(Svaasand and Ellingsen, (1983) Photochem Photobiol. 38:293-299).Altering the biologic interactions and physical characteristics of thephotosensitizer can alter these values. In general, photosensitizerswith longer absorbing wavelengths and higher molar absorptioncoefficients at these wavelengths are more effective photodynamicagents.

The light for photoactivation can be produced and delivered to the siteof infection or to a diagnostic reaction by any suitable means known inthe art. Photoactivating light can be delivered from a light source,such as a laser or optical fiber. Optical fiber devices that directlyilluminate the site of inflammation or a diagnostic reaction can deliverthe photoactivating light. For example, for therapeutic uses, the lightcan be delivered by optical fibers threaded through small gaugehypodermic needles. Light can be delivered by an appropriateintravascular catheter, such as those described in U.S. Pat. Nos.6,246,901 and 6,096,289, which can contain an optical fiber. Opticalfibers can also be passed through arthroscopes. In addition, light canbe transmitted by percutaneous instrumentation using optical fibers orcannulated waveguides. For open surgical sites, suitable light sourcesinclude broadband conventional light sources, broad arrays oflight-emitting diodes (LEDs), and defocused laser beams.

Delivery can be by all methods known in the art, includingtransillumination. Some photosensitizers can be activated by nearinfrared light, which penetrates more deeply into biological tissue thanother wavelengths. Thus, near infrared light is advantageous fortransillumination. Transillumination can be performed using a variety ofdevices. The devices can utilize laser or non-laser sources, (e.g.,lightboxes or convergent light beams).

In aspects where treatment is desired, the dosage of photosensitizercomposition, and light activating the photosensitizer composition, isadministered in an amount sufficient to produce a phototoxic species.For example, where the photosensitizer is chlorin_(e6), administrationto humans is in a dosage range of about 0.1 to about 10 mg/kg,preferably about 1 to about 5 mg/kg more preferably about 2 to about 4mg/kg and the light delivery time is spaced in intervals of about 30minutes to about 3 days, preferably about 12 hours to about 48 hours,and more preferably about 24 hours. The light dose administered is inthe range of about 2-500 J/cm², preferably about 5 to about 50 J/cm²,and more preferably about 5 to about 10 J/cm². The fluence rate is inthe range of about 20 to about 500 mw/cm², preferably about 50 to about300 mw/cm² and more preferably about 100 to about 200 mw/cm². There is areciprocal relationship between photosensitizer compositions and lightdose, thus, determination of suitable wavelength, light intensity, andduration of illumination is within ordinary skill in the art.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time, orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation.

Irradiation of the appropriate wavelength for a given compound for thetherapeutic or diagnostic methods of the invention may be administeredby a variety of wavelengths. Methods for irradiation include, but arenot limited to, the administration of laser, nonlaser, or broad bandlight. Irradiation can be produced by extracorporeal or intraarticulargeneration of light of the appropriate wavelength. Light used in theinvention may be administered using any device capable of delivering therequisite power of light including, but not limited to, fiber opticinstruments, arthroscopic instruments, or instruments that providetransillumination.

The wavelength and power of light can be adjusted according to standardmethods known in the art to control the production of phototoxic speciesor a fluorescence resonse. Thus, under certain conditions (e.g., lowpower, low fluence rate, shorter wavelength of light or some combinationthereof), a fluorescent species is primarily produced from thephotosensitizer and any reactive species produced has a negligibleeffect. These conditions are easily adapted to bring about theproduction of a phototoxic species. For example, where thephotosensitizer is chlorin_(e6), the light dose administered to producea fluorescent species and an insubstantial reactive species is less thanabout 10 J/cm, preferably less than about 5 J/cm and more preferablyless than about 1 J/cm. Determination of suitable wavelength, lightintensity, and duration of illumination for any photosensitizer iswithin the level of ordinary skill in the art.

In certain aspects directed to diagnostic methods, dequenched orphotoactivatable photosensitizers (e.g., following cleavage of linker)can be detected by illuminating the photosensitizers with suitablewavelength of light and then detecting the response (e.g., fluorescenceemission).

A sample can be illuminated with a wavelength of light selected to givea detectable optical response, and observed with a means for detectingthe optical response. Equipment that is useful for illuminating thepresent compounds and compositions of the invention includes, but is notlimited to, hand-held ultraviolet lamps, mercury arc lamps, xenon lamps,lasers and laser diodes. These illumination sources are opticallyintegrated into laser scanners, fluorescence microplate readers orstandard or microfluorometers, or any other suitable known means fordetecting and/or measuring the signal (e.g., a fluorescence signal).

The herein disclosed photosensitizers may, at any time after or duringan assay, be illuminated with a wavelength of light that results in adetectable optical response, and observed with a means for detecting andmeasuring the optical response. Upon illumination, such as by anultraviolet or visible wavelength emission lamp, an arc lamp, a laser,or even sunlight or ordinary room light, the fluorescent compounds,including those bound to the complementary specific binding pair member,display intense visible absorption as well as fluorescence emission.Selected equipment that is useful for illuminating the fluorescentcompounds of the invention includes, but is not limited to, hand-heldultraviolet lamps, mercury arc lamps, xenon lamps, argon lasers, laserdiodes, and YAG lasers. These illumination sources can be optionallyintegrated into laser scanners, fluorescence microplate readers,standard or mini fluorometers, or chromatographic detectors. Anysuitable computer software for measuring, processing and displayingimages and/or data pertaining to the process of detecting and measuringsignal sequences will be known to the skilled artisan and arecontemplated by the invention.

Fluorescence emissions can be optionally detected by visual inspection,or by use of any of the following devices: CCD cameras, video cameras,photographic film, laser scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, a fluorescence microscope or afluorometer, the instrument is optionally used to distinguish anddiscriminate between the fluorescent compounds of the invention and asecond fluorophore with detectably different optical properties,typically by distinguishing the fluorescence response of the fluorescentcompounds of the invention from that of the second fluorophore. Where asample is examined using a flow cytometer, examination of the sampleoptionally includes isolation of particles within the sample based onthe fluorescence response by using a sorting device.

The above description is not meant to limit the means, methods orinstrumentation that can be used to detect, measure, quantitate, andanalyze signals (e.g., fluorescence) produced by the photosensitizers ofthe invention in connection with those embodiments pertaining to thediagnostic methods of the invention. Any suitable means, methods orinstrumentation for detecting, measuring, quantitating and analyzingphotosensitizer signals are contemplated.

Targets and Samples

In certain embodiments, the photosensitizer compositions of theinvention can be used in therapeutic methods to treat a bacterialinfection in a subject in need thereof, e.g., where the infection is dueto an antibiotic resistant pathogen.

The subject can be a living animal or human (e.g., host) carrying anunwanted organism (e.g., pathogen), that is, an organism that is atarget for photodynamic therapy. The subject can be a mammal, such as ahuman or a non-human mammal (e.g., a dog, cat, pig, cow, sheep, goat,horse, rat, or mouse). The subject may be further immune deficient;presently or previously undergoing treatment for cancer (e.g., bychemotherapy or radiation therapy); or presently or previouslyundergoing antibiotic therapy or an immunosuppressive therapy.

In certain other aspects relating to use of the photosensitizercompositions of the invention for diagnostic purposes, e.g., detectionof and/or quantitation of a beta-lactamase activity, a sample can befrom any source, including samples obtained directly from infectedtissues or bodily fluids (e.g., blood, urine, feces, skin, lymph, spinalfluid, muscle, heart, brain, stomach, intestine, and any other organthat my be carrying an infection to be treated with antibiotics), orbacterial cultures obtained from natural sources (e.g., patients, soils,sediments) or from commercial or stock sources which are desired to beassayed or tested for antibiotic resistance.

In aspects relating to therapeutic uses, an organism that is targetedfor destruction by the methods and compositions described herein is anunwanted organism, unwanted in that it infects a host organism (or acell thereof) and causes or aggravates a disease or disorder in thathost.

Target organisms can be cellular. Such target organisms include at leasta boundary cell membrane and are capable of energy production, nucleicacid synthesis, and contain ribosomes and are capable of ribosomalprotein synthesis. Cells can be unicellular or multicellular, and saidunicellular organisms can be prokaryotic or eukaryotic. The target cellsmay express or produce an antibiotic resistance phenotype (e.g.,beta-lactamase phenotype) to be tested or evaluated using the herewithdisclosed methods.

Prokaryotic target organisms for treatment and/or diagnosis inaccordance with the method of the invention include bacteria, whichbacteria can be Gram negative or Gram positive, or which are lackingcell walls. The Gram stain basis of distinguishing bacteria, based onwhether or not cells of a specific strain or species of bacteria take upa stain, or are stained with the counterstain only, is known to those ofskill in the art.

Gram negative, largely β lactamase-producing, bacterial genera suitableas target organisms for treatment and/or diagnosis include Neisseria,Pasteurella, Proteus, Pseudomonas, Streptophomonas, Burkholderia,Acinetobacter, Serratia, Salmonella, Enterobacter, Escherichia,Haemophilus, and Klebsiella. Streptophomonas maltophilia, Burkholderiacepacia, and Acinetobacter baumannii are, for example, common colonizersof patients in an intensive care setting. Gram positive bacterial generasuitable as target organisms include Staphylococcus and Enterococcus.

Other bacterial pathogens to be contemplated herein as “unwantedorganisms” and, thus, to be targeted for destruction and or diagnosticanalysis, include, without limitation, Mycobacterium tuberculosis,Leishmania, Mycobacterium leprae, and Sheigella. Leishmania do notproduce β lactamase. Rather, they produce surface metalloproteinasegp63, which can, for example, cleave the heptapeptide AYLKKWV. Thus, thelatter polypeptide may serve as a suitable enzyme cleavage site within alinker in a photosensitizer composition according to the invention, foruse in the therapeutic and/or diagnostic methods of the invention.

In some therapeutic aspects, pathogens that can be targeted by thecompositions and methods of the present invention can be found on anylight-accessible surfaces or in light-accessible areas, for example, inhuman and animal subjects. In the cases of humans and animals,infections of the epidermis, oral cavity, nasal cavity, sinuses, ears,lungs, urogenital tract, and gastrointestinal tract are lightaccessible. Epidermal infections include subcutaneous infections,especially localized lesions, which infections are light-accessible.Infections of the peritoneal cavity, such as those resulting from burstappendicitis, are light accessible via at least laparoscopic devices. Avariety of skin infections which are refractory to antibiotics orlong-term antifungal treatment, for example, dermatophycoses of thetoenail, are suitable for photodynamic therapy using the methods andcompositions of the invention. In certain diagnostic methods of theinvention, samples comprising beta-lactamase enzymes or bacterialbeta-lactamase producers can be obtained from any of the above diseasedor infectious cells and/or tissues to identify and evaluate suitableantibiotic regimes that can be used in treatment.

Lung infection can occur with a variety of bacterial genera and species,which include the pseudomonads, which are the primary cause of death ofcystic fibrosis patients, Klebsiella, and can also occur with a varietyof virus strains. As pathogens of the lung are increasingly resistant toclassical antibiotic therapies, photodynamic therapy and/or diagnosiswith the compositions of the instant invention offer an alternativemethod for eliminating and/or diagnosing these unwanted organisms thatis independent of the microbial mechanisms of resistance.

Additional epidermal infections and infections of deeper tissues arisefrom burns, scrapes, cuts, and puncture wounds. In one aspect, PDTtreatment and/or diagnosis with the compositions of the instantinvention are useful for sterilization of such potential infectioussites, which can rapidly lead to toxic shock, a frequent concomitant ofbullet wounds, and for treating the sites to eliminate or reduceunwanted infectious organisms or determining a suitable and effectiveantibiotic regimen. A major cause of infection in wounds, especiallyburns, is the Gram negative aerobic bacterium Pseudomonas. This organismproduces an exotoxin which has been shown to retard wound healing.Multi-antibiotic resistant P. aeruginosa strains are becoming asignificant problem, especially in burns units of large hospitals.Pseudomonads also produce fulminating infections of the cornea.Escherichia coli along with Staphylococcus aureus are the two mostcommon bacteria in infected wounds.

Other sites of unwanted target organisms include the urogenital tract,the peritoneal cavity, the inner and outer ear, the nasal cavity and thegastrointestinal tract. Infectious sites of proliferation of unwantedtarget organisms in tissues of mesothelial and endothelial origin arealso accessible to PDT by minimally invasive techniques.

In other specific embodiments, areas of infection are notlight-accessible. Such areas can be accessed, for example, with the useof light-emitting probes or catheters. Thus, delivery of the light to arecessed, or otherwise inaccessible physiological location can befacilitated by flexible fiber optics (implicit in this statement is theidea that one can irradiate either a broad field, such as the lung or alobe of the lung, or a narrow field where bacterial cells may havelocalized). The source of the light needed to inactivate the compoundsof the invention can be an inexpensive diode laser or a non-coherentlight source.

The pathogens to be targeted by the diagnostic and/or therapeuticmethods of the invention using the compositions of the invention may benaturally or non-naturally occurring. Non-naturally occurring pathogenscomprise pathogens recombinantly engineered, for example, to exhibitresistance to certain standard antibodies. In a situation ofbioterrorism, for example, one might envision a pathogen that does notnaturally produce β lactamase being engineered to produce the latter.Recombinantly engineering a naturally occurring pathogen to exhibitmultiple antibody resistance would yield a highly virulent straindifficult to combat by standard treatment measures (such as penicillin).

These and other bacterial groups and genera not listed here will berecognized by the skilled artisan as suitable target bacteria for thecompositions of the invention. Thus, the above lists are used toillustrate applications of the present invention to major groups ofsuitable target organisms, but not to delimit the invention to thespecies, genera, families, orders or classes so listed.

The pathogen may be contained within a host cell, such as a phagocyte(e.g., a macrophage). Further, within that cell, the pathogen may becontained (wholly or partly) within a vacuole, vesicle, or organelle.

In aspects involving diagnostic uses of the photosensitizer compositionsof the inventions, biological samples can be obtained from any of theabove mentioned physiological locations for bacterial infection in orderto measure, assay, or evaluate the production of various enzymaticvirulence factors, e.g., beta-lactamase.

Antibacterial Compositions

In certain embodiments, the photosensitizer compositions of the presentinvention, or their pharmaceutically acceptable salts or esters, may beformulated into an antibacterial composition for treating targetbacterial infections in accordance with the invention. The antibacterialcompositions comprise one or more photosensitizer compositions as theactive ingredient(s), in association with an organic or inorganic, solidor liquid carrier suitable for oral administration or non-oraladministration or external applications, when said photosensitizercomposition is to be administered for the purpose of therapeuticallytreating bacterial infections, in particular, those bacterial infectionsthat are resistant to antibiotics.

This antibacterial composition may be prepared in the form of anyconventional formulations, which include capsules, tablets, ointments,suppossitories, solutions, suspensions, emulsions and so on. Ifnecessary, the above formulation may further contain a supplementaryagent, stabilizer, wetting agent or emulsifier, or buffering agent, orany of other conventional additives.

Thus, the antibacterial composition of the invention may be administeredin the form of a formulation, such as intravenous or intramuscularinjections, orally administrable preparations, suppositories or thelike. The excipient or carrier present in the composition may be chosenfrom the pharmaceutically acceptable ones, and the sort of the excipientor carrier varies depending on the route of administration and themethod of administration. For instance, a liquid carrier may be used,which can include water, ethanol, or animal and vegetable oils, such assoybean oil, sesame oil, or mineral oil or synthetic oil, and so on. Asa solid carrier, sugar, such as maltose and sucrose, an amino acid, suchas lysine, a cellulose derivative, such as hydroxypropylcellulose andthe like, a polysaccharide, such as cyclodextrins, and an organic acidsalt, such as magnesium stearate, and the like.

When the antibacterial composition is formulated into an injection, ingeneral, the carrier may desirably be physiological saline, variousbuffered solutions, aqueous solutions of a sugar such as glucose,inositol, mannitol and the like, or a glycol such as ethylene glycol,polyethylene glycol and the like. Further, the anti-bacterialcomposition may also be formulated into a lyophilised preparation inassociation with an excipient which may be a sugar such as inositol,mannitol, glucose, mannose, maltose, sucrose and the like, or an aminoacid such as phenylalanine and the like. Upon administration, thelyophilised preparation may be dissolved into a solvent suitable for theinjection, for example, a liquid available for intravenous injection,which may be sterile water, physiological saline, aqueous solution ofglucose, solution of electrolytes and aqueous solution of amino acids,and the like.

The proportion of the photosensitizer composition formulated as anantibacterial composition may vary according to the type of theformulation but usually may be 0.1 to 99% by weight, preferably 1 to 90%by weight of the composition. For instance, an injectable solution maynormally contain 0.1 to 10% by weight of the active ingredient compound.When the anti-bacterial composition is to be given orally, it is used inthe form of a preparation such as tablets, capsules, powders, granules,dry syrups, liquids, syrups and the like, in association with a solidcarrier or a liquid carrier as mentioned in the above. For the capsules,tablets, granules and powders, in general, the proportion of the activeingredient compound present therein may be 3 to 99% by weight,preferably 5 to 90% by weight of the composition, with the balance beingthe carrier.

The dosage of the photosensitizer composition of the invention to beused as the active ingredient, or its salt or ester depends on the age,body weight and symptoms of patients, and the purposes of thetherapeutic treatment, and other factors. The dosage is to give aneffective amount of the photosensitizer composition to combat againstthe infecting bacteria. The photosensitizer composition at a necessarydosage may be administered continuously or intermittently as long as atotal dosage of the photosensitizer composition does not exceed aspecific level which is decided in view of the results of animal testsand various circumstances.

When administered parenterally, the total dosage of the photosensitizercomposition of this invention is, of course, administered withappropriate adjustments being done in view of the way of administration,the conditions of patients such as age, body weight and sex, as well asfoods and medicines concurrently administered. Suitable dosage andadministration frequency of the photosensitizer composition of thisinvention under given conditions can be determined by expert physicianthrough the tests of determining optimal dosage in light of theabove-mentioned guidelines. These guidelines for administration alsoapply to oral administration of the photosensitizer composition of theinvention.

Diagnostic Methods and Kits

In yet another aspect, the present invention provides methods anddiagnostic kits for using the photosensitizer compositions describedherein for a wide array of diagnostic applications relating generally tothe qualitative and/or quantitative detection of enzymes involved inantibiotic resistance, such as, beta-lactamase enzymes. In some aspects,the photosensitizer molecules of the invention are quenched prior tocleavage of the linker joining them together. Once the linker iscleaved, however, the photosensitizer molecules become physicallyseparated, upon which time, they become activatable such that, whenactivated, they produce a detectable signal, e.g., a fluorescencesignal. This feature can be utilized in accordance with the inventionfor qualitative and/or quantitative detection and/or measurement of theenzyme utilizing the linker as substrate, i.e., the “the activity ofinterest,” e.g., a beta-lactamase. Such methods can also be used toobtain information regarding the substrate specificity of the activityof interest, e.g., a beta-lactamase activity, which can aid in thedetermination of an appropriate antibiotic therapy can be implemented.

The enzymes that are tested using the methods of the invention can befrom any organism of interest, either isolated from an infection ordirectly from a cell or tissue, or those organisms that are isolatedfrom the body and kept in culture or storage etc. The inventive assayscan also be directed against the enzymes themselves, either in purifiedor non-purified form. The enzymes can be from any organism, for example,a Gram negative bacterium, such as, β lactamase-producing bacteria,including Neisseria, Pasteurella, Proteus, Pseudomonas, Streptophomonas,Burkholderia, Acinetobacter, Serratia, Salmonella, Enterobacter,Escherichia, Haemophilus, and Klebsiella. Streptophomonas maltophilia,Burkholderia cepacia, and Acinetobacter baumannii are, for example,common colonizers of patients in an intensive care setting. Grampositive bacterial genera suitable as target organisms includeStaphylococcus and Enterococcus.

In one aspect, the present invention provides a method for detecting thepresence of an enzyme activity of interest, e.g., a beta-lactamaseactivity, in a sample. The method includes contacting the sample with atleast one photosensitizer composition described herein, andphotoactivating the sample to induce a signal to be released fromunquenched photosensitizers, and detecting the signal, e.g., afluorescence signal.

The “signal” as used herein refers to a detectable response produced byphotoactivation of the unquenched photosensitizers of the inventionwhich is directly or indirectly detectable (observable) either by visualobservation or by instrumentation or other suitable means for detection.Typically, the detectable response is a detectable response in anoptical property, such as a change in the wavelength distributionpatterns or intensity of absorbance or fluorescence or a change in lightscatter, fluorescence lifetime, fluorescence polarization, or acombination of such parameters in a sample. The signal may occurthroughout the sample or in a localized portion of the sample. Thepresence or absence of the signal after the elapsed time is indicativeof one or more characteristics of the sample. Comparison of the amountof the compound of the invention with a standard or expected responsecan be used to determine whether and to what degree a sample possessesthe enzyme (and enzymatic activity) of interest.

In one aspect, the present invention provides methods for detecting anenzyme activity of interest, e.g., beta-lactamase activity, from asample by use of the photosensitizer compositions of the invention. Suchdetection methods can be per se detection methods, which, as usedherein, refers to a qualitative, ‘yes/no,’ detection of a an enzymeactivity of interest, e.g., beta-lactamase activity. In advantageousaspects, the methods of the invention provide for per se detectionmethods to detect beta-lactamase activity in a sample, wherein thedetection of activity against a photosensitizer composition of theinvention indicates a resistance to the particular antibiotic-basedlinker because said linker is cleaved by the activity. In other aspects,information regarding the susceptibility of a linker of the invention toa bacterial enzyme, e.g., a beta-lactamase enzyme from a biologicalsample, can be used to determine an effective antibiotic regimen to beapplied against an infection. In some aspects, the antibiotic regimencan comprise the administration of an antibiotic which has a differentstructure than that of the cleaved linker. In still other aspects,information regarding linker susceptibility can be used to determine thetype or class of beta-lactamase enzyme produced by the bacteria causingan infection. Beta-lactamase types or classes are discussed hereinabove. It will be understood that knowledge of the type or class of thebeta-lactamase enzyme of a pathogenic organism can provide informationregarding the particular antibiotics that might be effective against thepathogen of interest.

In other aspects, the detection methods of the invention provide a meansfor detecting an enzyme activity of interest, e.g., beta-lactamaseactivity, as a function of another process that involves production ofthat particular enzyme having the activity. In a particular aspect, theinvention provides a method of detecting a beta-lactamase activity as afunction of the production of said enzyme due to another process (suchas where a beta-lactamase encoding nucleic acid is used as a reportergene to measure expression of the nucleic acid). Such detection methodsmay be practiced on both cell-free and cellular systems (e.g.,intracellular detection). Examples of methods for detectingbeta-lactamase activity in which the presently disclosed photosensitizercompositions of the invention may be utilized as substrates for abeta-lactamase include those methods disclosed in U.S. Pat. Nos.5,955,604, 5,741,657, 6,031,094, 6,291,162, and 6,472,205, each of whichare incorporated herein by reference.

As described in the above-referenced United States patents (such as,U.S. Pat. No. 6,472,205), cells to be assayed for beta-lactamaseactivity may be contacted with a photosensitizer composition describedherein. In the presence of a beta-lactamase, the linker substrate iscleaved, rendering the photosensitizers to transition into an unquenchedstate, i.e., activatable state. Upon illumination with the appropriatewavelength and/or quantity of light, a detectable signal is produced,such as the generation of fluorescence emissions. If a beta-lactamase ispresent in the sample, and the linker is susceptible to being cleaved bythe said enzyme, then the sample will exhibit increased fluorescencewhen contacted with a photosensitizer composition of the invention. Suchfluorescence changes can be detected by exciting the sample withradiation of a first wavelength, which excites the photosensitizergroup, which emits radiation of a second wavelength, which can bedetected. The amount of the emission is measured, and compared to propercontrol or background values. The amount of emitted radiation thatdiffers from the background and control levels, either increased ordecreased, correlates with the amount or activity of the beta-lactamasein the sample. Standard curves can be determined for quantitativemeasurements.

In a further aspect, the present invention provides a method fordetermining whether an enzyme of interest in a sample, e.g.,beta-lactamase enzyme, can cleave a disclosed linker of aphotosensitizer composition of the invention. The method involvescontacting the sample with a photosensitizer composition of the presentinvention, thereby leading to the cleavage of any suspectible linkers,followed by exciting the sample with radiation of one or morewavelengths that are suitable for the cleaved compound, and determiningthe degree of fluorescence emitted from the sample. A degree offluorescence emitted from the sample that is greater than an expecteddegree (or baseline level) indicates that the beta-lactamase enzyme cancleave the compound and that the compound is a substrate for thebeta-lactamase enzyme.

In another aspect, a method for determining whether a sample containsbeta-lactamase activity is provided. The method involves contacting thesample comprising a beta-lactamase activity with a photosensitizercomposition of the invention under conditions sufficient to allow thecleavage of any susceptible linker moiety by the beta-lactamaseactivity. Cleavage of a linker causes the photosensitizers to separateand become unquenched and consequently, photoactivatable. The sample isthen irradiated with one or more wavelengths that are absorbed by theunquenched photosensitizers, which then emit fluorescence of aparticular wavelength and strength. A degree of fluorescence emittedfrom the sample that is greater than expected, i.e., greater than abaseline control sample containing no beta-lactamase activity, indicatesthe presence of beta-lactamase activity in the sample. One aspect ofthis method is for determining the amount of an enzyme in a sample bydetermining the degree of fluorescence emitted at a first and secondtime after contacting the sample with a compound of the presentinvention. The difference in the degree of fluorescence emitted from thesample at the first and second time is determined, and the differencereflects the amount of a beta-lactamase enzyme in the sample.

In another aspect, the present invention provides screening assays thatutilize the disclosed photosensitizer compositions and a host cell, suchas a mammalian cell, transfected with at least one recombinant nucleicacid molecule encoding at least one protein having beta-lactamaseactivity. Such recombinant nucleic acid molecules can include expressioncontrol sequences adapted for function in a eukaryotic cell, such as avertebrate cell, operatively linked to a nucleotide sequence coding forthe expression of a beta-lactamase enzyme.

In yet another aspect, methods are provided for determining the amountof beta-lactamase activity in a cell. This method involves contacting asample, including a host cell that is transfected with a recombinantnucleic acid molecule that includes a nucleic acid sequence coding forthe expression of a beta-lactamase. The sample can comprise whole hostcells, or an extract of the host cells, which is contacted with aphotosenstitizer composition of the present invention. The amount ofphotosenstitizer composition cleaved is measured by measuring adetectable response, whereby the amount of substrate cleaved is relatedto the amount of beta-lactamase activity in the host cell.

In another aspect, a method for monitoring the expression of a geneoperably linked to a set of expression control sequences is provided.The method involves providing a host cell transfected with a recombinantnucleic acid molecule, where the nucleic acid molecule comprises a setof expression control sequences operatively linked to nucleic acidsequences coding for the expression of a beta-lactamase enzyme, exceptif the host cell is a fungus, the beta-lactamase is a cytosolicbeta-lactamase enzyme. A sample comprising the host cell, or an extractor conditioned medium produced therefrom or thereby, is contacted with adisclosed compound. The amount of compound cleaved is determined,wherein the amount of substrate cleaved is related to the amount ofbeta-lactamase activity in the host eukaryotic cell, which is related tothe expression of the gene.

In another aspect, a method is provided for determining whether a testcompound alters the expression of nucleic acid sequence operably linkedto an expression control sequence(s). The method involves contacting ahost cell transfected with a recombinant nucleic acid sequence, wherethe recombinant nucleic acid comprises an expression control sequence(s)operably linked to a nucleic acid sequence coding for a beta-lactamase.The host cell is contacted with the test compound, and the host cell isthen contacted with a disclosed photosenstitizer composition. The amountof the photosenstitizer composition cleaved is then measured, wherebythe amount of the photosenstitizer composition cleaved is related to theamount of beta-lactamase activity in the cell. In addition, the amountof photosenstitizer composition cleaved in the presence of the testcompound can be compared to the amount of photosenstitizer compositioncleaved in the absence of the test compound to determine if the testcompound alters expression regulated by the control sequence.

In another aspect, a method for clonal selection is provided, whereincells that are presumably transfected with a recombinant nucleic acidmolecule comprising a sequence coding for a beta-lactamase are contactedwith a disclosed compound. Those cells that are in fact transfected withthe recombinant nucleic acid molecule will exhibit beta-lactamaseactivity, which is detected by measuring the detectable optical changeproduced upon cleavage of the photosenstitizer composition. Cells thatexhibit beta-lactamase activity, or greater than a predetermined levelof beta-lactamase activity may be selected, and propagated if desired.Selection of cells exhibiting beta-lactamase activity can beaccomplished using fluorescence activated cell sorting (FACS), using,for example, a Becton Dickinson FACS Vantage.

Another aspect is to use a beta-lactamase reporter gene and a compoundof the present invention to screen test chemicals for biochemicalactivity. A cell transfected with a recombinant nucleic acid moleculethat includes at least one expression control sequence operably linkedto at least one nucleic acid sequence encoding for the expression of abeta-lactamase enzyme is contacted with a test chemical. The cell isthen contacted with a disclosed photosenstitizer composition and theamount of the photosenstitizer composition cleaved is measured. Theamount of photosenstitizer composition cleaved reflects the amount ofbeta-lactamase activity within the at least one cell, and reflects thebiochemical activity of the test chemical. The amount ofphotosenstitizer composition cleaved in the presence of the testchemical is compared to the amount of photosenstitizer compositioncleaved in the absence of the test chemical to determine if the testchemical increases, decreases or does not alter expression under controlof the control sequence.

The interaction of a particular disclosed photosensitizer compositionwith a particular beta-lactamase enzyme can be readily determined. Inone embodiment, such a method involves contacting the sample with aphotosensitizer composition to cause the cleavage of the linker,exciting at one or more wavelengths that are suitable for the unquenchedphotosensitizers, and determining the degree of fluorescence in thesample. A degree of fluorescence that is greater than an expected amountin the absence of beta-lactamase activity indicates that the particularbeta-lactamase enzyme can cleave the particular compound. The amount offluorescence expected can be determined using, for example, a controlsample, or control values determined contemporaneously, prior to, orafter a particular assay was performed. Such expected values can includea statistical analysis, such as a mean and standard deviation, toprovide a chosen statistical confidence level. Both naturally occurringbeta-lactamase enzymes and beta-lactamase enzymes prepared bymutagenesis can be tested with a particular disclosed compound.

Any of the above methods specifically disclosed, and other method thatinclude the use of the disclosed photosenstitizer compositions to detectbeta-lactamase activity may further include use of the methods describedin U.S. Pat. No. 6,284,461 to increase the signal to noise ratio of thedisclosed assays.

In addition, the disclosed compounds may be used to detectbeta-lactamase activity in a wide variety of biologically importantenvironments, such as human blood serum, the cytoplasm of cells andintracellular compartments, which can facilitate the measurement ofperiplasmic or secreted beta-lactamase enzyme. In addition, the presence(for example, in human serum, pus, urine, or other fluid, sample, ortissue) of bacteria resistant to beta-lactam antibiotics may be readilydetected by using the disclosed compounds. Only in the presence of anactive beta-lactamase enzyme is there a fluorescence spectrum that ischaracteristic of the photosensitizers. Such methods include contactingthe environment with a disclosed photosenstitizer composition anddetecting any beta-lactamase activity present by measuring thedetectable optical change that occurs upon cleavage of thephotosenstitizer composition by a beta-lactamase. Further, theexpression of any target protein may be detected by fusing a geneencoding the target protein to a beta-lactamase gene, which can belocalized by immunostaining or fluorescence or electron microscopy. Forexample, beta-lactamase fusion proteins can be detected in the lumen oforganelles through the use of the substrates of the invention. In thisinstance, only subcellular compartments containing the fusion proteinfluoresce at a wavelength characteristic of the cleaved substrate,whereas all others fluoresce at a wavelength characteristic of theintact molecule.

In yet another aspect, the present invention provides a method ofevaluating the substrate specificities of various beta-lactamases, i.e.,typing a beta-lactamase in one of the known classes of beta-lactamaseenzymes (e.g., type A, B, C, or D). In one aspect, the substratespecificity of a beta-lactamase can be determined by contacting a seriesof photosensitizer compositions comprising a plurality of differentlinkers. The linkers of the series of photosensitizer compositions ofthe invention can be any of the herein disclosed linkers, e.g.,cephalosporin, penicillin, penem, a carbapenem or a moncyclic mobactem,along with numerous others, including any fragments and/or derivativesthereof. Linkers that are susceptible to cleavage represent thosecompounds against which the beta-lactamase shows substrate specificity.Linkers that are not susceptible to cleavage represent those compoundsagainst which the beta-lactamase enzymes are not effective and which donot have substrate specificity. Those linkers showing substratespecificity represent those antibiotics which would be not be effectivein treating an infection caused by an organism expressing suchbeta-lactamases. In this way, an appropriate antibiotic regime can bedesigned and which would reflect the particular substrate specificity ofthe encoded beta-lactamase of the infective organism. Thus, ineffectiveantibiotics can be avoided.

Samples may be obtained and/or prepared from any suitable source usingany suitable means for preparation. Samples include, without limitation,any biological material that is thought to contain an enzyme activity ofinterest, e.g., a beta-lactamase. The enzyme activity of interest isadvantageously an enzyme which confers antibiotic-resistance in abacterium (e.g., beta-lactamase). Alternatively, samples also includematerial in which a beta-lactamase has been added. The samples can be abiological fluid, such as whole blood, plasma, serum, nasal secretions,sputum, saliva, urine, sweat, transdermal exudates, cerebrospinal fluid,or the like. Biological fluids also include tissue and cell culturemedium wherein an analyte of interest has been secreted into the medium.Alternatively, the sample may be whole organs, tissue or cells from theanimal. Examples of sources of such samples include muscle, eye, skin,gonads, lymph nodes, heart, brain, lung, liver, kidney, spleen, thymus,pancreas, solid tumors, macrophages, mammary glands, mesothelium, andthe like. Cells include without limitation prokaryotic cells andeukaryotic cells that include primary cultures and immortalized celllines. Eukaryotic cells include without limitation ovary cells,epithelial cells, circulating immune cells, beta cells, hepatocytes, andneurons.

In many instances, it may be advantageous to add a small amount of anon-ionic detergent to the sample. Generally the detergent will bepresent in from about 0.01 to 0.1 vol. %. Illustrative non-ionicdetergents include the polyoxyalkylene diols, e.g. Pluronics, Tweens,Triton X-100, etc.

Kits

In another aspect, the present invention provides a kit that includesone or more of the disclosed photosensitizer compositions. The kit canalso include an additional component, for example, instructions forusing the photosensitizer compositions in one or more methods,additional molecules (such as a beta-lactamase, or a nucleic acid codingfor a beta-lactamase such as a vector having a beta-lactamase sequenceas a reporter), substances (such as a reaction buffer), or biologicalcomponents (such as cells, or cell extracts). For example, cells (e.g.,prokaryotic or eukaryotic cells) which contain beta-lactamase activityand/or at least one beta-lactamase substrate, as well as compositionsand reaction mixtures which contain such cells can be included in thekits. Cells may further include receptor and signaling molecules thatregulate expression of nucleic acid sequences within the cell, eithersequences found on vectors, or in the nucleus or mitochondria of thecells. Cells, compositions and reaction mixtures that include at leastone of the disclosed compounds are also part of the disclosure,regardless of whether or not they are part of a “kit” per se.

In some aspects, the kit includes a solid support covalently bonded to adisclosed photosenstitizer composition and instructions for detecting abeta-lactamase in a sample with the solid support. In other aspects, thekit includes a disclosed photosenstitizer composition that includes areactive group, a solid support and instructions which specify how toimmobilize the compound on the solid support and how, after forming theimmobilized photosenstitizer composition, to detect a beta-lactamase.Alternatively, the kit includes a solid support bearing reactive groupsthat can react with and immobilize a beta-lactamase, and instructionsthat specify how to immobilize beta-lactamases to the solid support andto detect such immobilized beta-lactamases using one or more of thephotosenstitizer compositions of the disclosure. Methods of detectingimmobilized beta-lactamases are presented above.

In another aspect, the kit may include compositions for the quantitativedetermination of a beta-lactamase in a sample. In an aspect, thecomposition comprises a sample containing a known amount of abeta-lactamase (such as a solution containing the known amount ofbeta-lactamase or cells expressing known amounts of the beta-lactmase)and a disclosed photosenstitizer composition of the invention, whereinthe photosenstitizer composition reacts with a beta-lactamase to producea detectable optical response that is proportional to the amount of thebeta-lactamase in the sample, for example, an amount of a fluorescentproduct or fluorescence emission that is proportional to the amount ofthe beta-lactmase in the sample.

Beta-lactamases that may be included in a kit according to thedisclosure can be of any type, and include both naturally-occurringbeta-lactmases and non-naturally-occurring beta-lactamases, such asthose disclosed in Bush et al. (1995) Antimicrob. Agents Chemother.39:1211-1233, which is incorporated herein by reference.

Those skilled in the art will appreciate that the polypeptides havingbeta-lactamase activity disclosed herein may be altered by, for example,mutating, deleting, and/or adding one or more amino acids and may stillbe used in the practice of the invention so long as the polypeptideretains detectable beta-lactamase activity toward at least one disclosedcompound. An example of a suitably altered polypeptide havingbeta-lactamase activity is one from which a signal peptide sequence hasbeen deleted and/or altered such that the polypeptide is retained in thecytosol of prokaryotic and/or eukaryotic cells.

In yet another aspect, the present invention provides a method and kitfor typing or characterizing a beta-lactamase enzyme in terms of itssubstrate specificity. In this embodiment, the kit comprises one or morephotosensitizer compositions disclosed herein. The kit further comprisesone or more competing beta-lactam antibiotics or derivatives thereof,such as any of those beta-lactam antibiotics described herein. Inpractice, the kit can be used generally to compare the level of signalgenerated by reaction of the one or more photosensitizer compositionswith the beta-lactamase enzyme of the kit in the presence and absence ofthe one or more competing beta-lactam antibiotics (or fragments orderivatives thereof). By comparing the signals generated with andwithout the one or more competing beta-lactam antibiotics, the skillartisan can determine the substrate specificity of the enzyme. It wouldbe expected that a reduced signal (relative to a baseline of aphotosenstitizer composition without a competing beta-lactam) of thephotosensitizer composition of the invention when in the presence of acompeting beta-lactam antibiotic suggests that the competing beta-lactamantibiotic is a cleavable substrate of the beta-lactamase, and as suchwould not be effective against a bacterium that expresses the testedbeta-lactamase. On the other hand, it would be expected that anunaffected or unchanged signal (relative to a baseline of aphotosensitizer composition without a competing beta-lactam) of thephotosensitizer composition of the invention when in the presence of acompeting beta-lactam antibiotic suggests that the competing beta-lactamantibiotic is not a cleavable substrate of the beta-lactamase, and assuch would be effective against a bacterium that expresses the testedbeta-lactamase.

Thus, in one aspect, the present invention provides a method and/or kitthat allows for the typing and/or classification of a beta-lactamase interms of substrate specificity with instructions for: performing anon-competitive reaction comprising the steps of (a) contacting thesample with a photosensitizer composition comprising a plurality ofphotosensitizers that are conjugated to a linker, wherein the linkercomprise a cleavage site for a beta-lactamase and wherein thephotosensitizers are in a quenched state; (b) cleaving the linker todequench the photosensitizers; (c) light-activating the composition toproduce a fluorescence signal; and (d) quantifying the fluorescencesignal with a detector.

EXAMPLES Example 1 Preparation of a Photosensitizer CompositionComprising a Polymer, β-Lactam Moiety and Photosensitizer

In one approach, the synthesis of the conjugates is based oncephalosporin, a commonly used β-lactam. It is conceivable to developpenem or carbapenem derivatives subsequently.

In the following, the photosensitizer (a porphyrin molecule with atleast one propionic side chain) is represented by PS—CH₂—CH₂—COOH. Thepolymer used in the synthetic routes shown below is a linear or branchedpoly(ethylene glycol) with propionic acid groups (PEG-CH₂—CH₂—COOH)(Senter, P. D., et al. (1995) Bioconjug. Chem. 6:389-394). However, thechemistry is applicable to similar polymeric materials containingavailable carboxylic side chains. In order to be released upon enzymatichydrolysis, the porphyrin molecule is advantageously linked at the3′-position of the cephalosporin. The cephalosporin-porphyrin moietyobtained can then be conjugated to the polymer using the amino group onthe β-lactam ring.

The preparation of three different conjugates is proposed, where theporphyrin and cephalosporin are linked via an ester:

or via a carbamate group:

The preparation of a cephalosporin-prophyrin ester comprises thefollowing steps:

A. Protection of the amino-group in the β-lactam ring

There are several ways to protect the amino group. One is representedbelow (Hanessian, S., et al. (1993) Can. J. Chem. 71:896-906):

Protected cephalosporin derivatives are commercially available. Otherprotecting groups include (Albrecht, H. A., et al., (1990) J. Med. Chem.33:77-86; Albrecht, H. A., et al. (1991) J. Med. Chem. 34:2857-2864;Alexander, R. P., et al. (1991) Tetrahedron Lett. 32:3269-3272):

-   -   For example, the following molecule (which comes with a        protected amino group) is called cephalothin.

B. Binding of the porphyrin at the 3′-position of the cephalosporin viaan ester function

-   -   i. Through a diazomethyl intermediate (Mobashery, S., et        al. (1986) J. Biol. Chem. 261:7879-7887)

In this scheme, pNBz=para-nitro-benzyl.

-   -   ii. Through a halogenated intermediate (Mobashery, S., et        al. (1986) J. Biol. Chem. 261:7879-7887)

-   -   iii. Through a hydroxymethyl intermediate (Hanessian, S., et        al. (1993) Can. J. Chem. 71:896-906)

C. Deprotection of the amino-group in the B-lactam ring (Albrecht, H.A., et al. (1991) J. Med. Chem. 34:669-675)

Deprotection of the amino group is also very often carried out usingPenicillin-G amidase (PGA) (Vrudhula, V. M., et al. (1995) J. Med. Chem.38:1380-1385).

D. Conjugation of the cephalosporin-porphyrin moiety to a polymer(Senter, P. D., et al. (1995) Bioconjug. Chem. 6:389-394)

The preparation of a cephalosporin-porphyrin carbamate comprises thefollowing steps:

A. Protection of the amino-group in the β-lactam ring (see above)

B. Binding of the porphyrin at the 3′-position of the cephalosporin viaa carbamate

-   -   i. Direct coupling between the porphyrin and cephalosporin        (Alexander, R. P., et al. (1991) Tetrahedron Lett. 32:3269-3272;        Rodrigues, M. L., et al. (1995) Chem. & Biol. 2:223-227;        Smith, K. M., et al. (1987) Heterocycles 26:1947-1963)

-   -   ii. Coupling through a linker (Alexander, R. P., et al. (1991)        Tetrahedron Lett. 32:3269-3272; Rodrigues, M. L., et al. (1995)        Chem. & Biol. 2:223-227; Boutorine, A. S., et al. (1996) J. Am.        Chem. Soc. 118:9469-9476)

C. Deprotection of the amino-group in the β-lacatam ring (see above)

D. Conjugation of the cephalosporin-porphyrin moiety to a polymer(Senter, P. D., et al. (1995) Bioconjug. Chem. 6:389-394) (see above)

Of additional note, if, after these chemical modifications, thecephalosporin derivatives described above retain their properties assubstrates for β-lactamases, one can expect to observe theenzyme-dependent release of three different porphyrin moieties:PS-Ch₂—CH₃,

PS—CH₂—CH₂—NH₂,

and PS—CH₂—CH₂—CO—NH—(CH₂)₄—NH₂:

Example 2. Development of Carbamate-Linked Photosensitizer, Inactive(with or without Light) while Linked and Light-Activatable Only whenReleased by the β-Lactamase Enzyme-Mediated Cleavage

Unlike conventional antibiotics, where hydrolysis of the beta-lactamring by β-lactamases causes inactivation, the beta-lactam ring openingof the prodrugs releases the photosensitizer and make itlight-activatable for photokilling (FIG. 1).

Synthesis

Commercially available 7-aminochephalosporanic acid was reacted withphenylacetyl chloride under Shotten-Baumann reaction conditions toachieve an amino protected chephalosporin molecule. This was furtherde-esterified using tetrabutylammonium hydroxide as a base to yieldeasily functionalizable hydroxy end group on cephalosporin. The laststep of the synthesis was achieved in a one-pot reaction sequence.Toluidine Blue O (TBO) was converted into its isocynate derivative inthe presence of diphosgene. The carbamate-linked prodrug (hereinsometimes referred to as prodrug 1) was obtained by adding Cephalosporinderivative to the same reaction mixture.

Synthesis of 7-[(2-phenylacetyl)amino] cephalosporanic acid

To a stirred mixture of sodium bicarbonate (2.1 g, 25 mmol) in water (40ml) and acetone (30 ml), added 7-(phenylacetyl)amino cephalosporanicacid. Stirred this solution for nearly 15 min in ice bath and slowlyadded phenylacetyl chloride (2.5 ml, 20 mmol) over the period of 30 min.This reaction mixture was stirred overnight and acidified to pH 2.0 withIN hydrochloric acid. Precipitates obtained were extracted withdichloromethane and washed with water. Dried over magnesium sulphate andsolvent evaporated to give off-white solid. The solid sample was stirredovernight in diethyl ether and filters to obtain crude product in 80%yield.

Synthesis of 7-[(2-phenylacetyl)amino] 3-hydrodxymethy cephalosporanicacid

To a suspension of 7-[(2-phenylacetyl)amino] cephalosporanic acid (0.5g, 1.28 mmol) in a a mixture of methane (4 ml) and water (2.5 ml),triethylamine (0.21 ml, 1.54 mmol) was added in 15 min at 0-5° C. Tothis solution, tetrabutylammonium hydroxide (30% solution in water, 1.53g, 1.92 mmol) was added at −18° C. in 30 minutes. The reaction mixturewas maintained at −18° C. for nearly 7.0 h and acidified to pH 5.0 usingglacial acetic acid. Purification was done using C-18 reverse phasecolumn and pure product was obtained as white solid in 67% yield.

Synthesis of Cephalosporanic Acid-Toluidine blueO Prodrug

To a magnetically stirred suspension of toludine blue O (0.1 g, 0.33mmol) in anhydrous THF (3 ml) under nitrogen was added a solution oftricholoromethyl chloroformate (19.7 μl, 0.164 mmol) over activatedcharcoal as a catalyst. The reaction mixture was stirred at 55° C. for30 min. Progress of reaction was monitored using mass spectroscopy forformation of isocynate derivative of toludine blue O. Cooled the flaskto room temperature and added a solution of 7-[(2-phenylacetyl)amino]3-hydrodxymethy cephalosporanic acid (0.15 g, 0.33 mmol) in anhydrousdichloromethane (1 ml). The reaction flask was cooled to 0° C. andslowly added diisopropylethylamine (57.0 μl, 0.33 mmol). Stirred for 3.0h and purified using C18 column with acetonirile and water as elutingsolvents. Pure product obtained as a blue solid in 25% yield.

¹H NMR spectra were obtained for 7-[(2-phenylacetyl)amino]cephalosporanic acid in CDCl₃ as a solvent, as well as for7-[(2-phenylacetyl)amino] 3-hydrodxymethy cephalosporanic acid inDMSO-d₆ as a solvent (FIG. 2). MS spectra were obtained for7-[(2-phenylacetyl)amino] 3-hydrodxymethy cephalosporanic acid andcephalosporanic acid-toluidine blue O prodrug (FIG. 3).

UV-visible spectra revealed blue shift in the absorption spectra of theprodrug, indicating extended conjugation, as well as quenching, ofcarbamate linked TBO photosensitizer (FIG. 4). Fluorescence spectrarevealed nearly an 8-fold reduction in fluorescence emission maxima at635 nm excitation, indicating quantitative quenching of thephotosensitizer upon conjugation with the cephalosporin moiety (FIG. 5).

Enzyme-Mediated Cleavage of the Prodrug

The prodrug obtained was further studied for release of photosensitizerin presence of β-lactamase from Enterobacter cloacae. For thefluorescence emission study of the prodrug, the solvent employed waswater, and the excitation wavelength 635 nm in the presence ofbeta-lactamase enzyme (from Enterobacter cloacae). Time-dependentfluorescence emission was also measured for photosensitizer release fromthe prodrug in the presence of enzyme. The results indicate an easyrelease and nearly 5-fold increase in excited stated properties withinminutes of incubation of prodrug with enzyme (FIG. 6).

Thus, the prodrug was synthesized and characterized. Furthermore, theprodrug showed quantitative quenching of the photosensitizer in theconjugated form. Additionally, the product demonstratedlactamase-specific activity.

Example 3 Construction and Use of a Beta-Lactamase Enzyme ActivatedPhotosensitizer Prodrug (β-LEAPP) Background

Photodynamic therapy (PDT) is an emerging approach for the treatment ofantibiotic resistant bacterial infections (Hamblin et al., 2004,Photochem. Photobiol. Sci., 3:436-50; Wainwright, J. Antimicrob.Chemother., 1998, 42:13-28). There are four fundamental constituents ofPDT: light, a photosensitizer, oxygen and a target. Photosensitizers(PS) are dyes that absorb light energy and transfer that energy to arecipient molecule thus producing reactive molecular intermediates thatdestroy the biological target (Hamblin et al., 2004, Photochem.Photobiol. Sci., 3:436-50). The effective doses required for theinactivation of bacteria via PDT can damage the surrounding host tissue(Gad et al., Photochm. Photobiol. Sci., 2004, 3:451-8). The developmentof more specific photosensitizers for the PDT of bacterial infectionswould reduce damage to host tissue and enhance the antibacterial effect.This example also demonstrates that such compounds are useful for thedetection, quantitation and typing of a target virulence enzyme (e.g.beta-lactamase) and may advantageously be used to determine anappropriate course of antibiotic therapy.

The present invention involves, in one aspect, the use of a substratefor a hydrolytic bacterial virulence enzyme (e.g., beta-lactamase) as alinker (e.g., beta-lactam ring) for two photosensitizers orfluorophores. The proximity of the linked fluorophores to each otherresults in a quenched (non-photoactive) state (see FIG. 7).

Upon hydrolysis of the substrate, the fluorophores are released fromquenching and become photoactive, capable of the absorption of lightenergy and the transfer of that energy to another molecule or therelease of that energy in the form of light fluorescence (see FIG. 7).

The photoactive phenothiazines are a group of tricyclic PSs that absorbin the red region of the electromagnetic spectrum (Cincotta et al.,Photochem. Photobiol., 1987, 46:751-8). EtNBS is abenzo[a]phenthiazinium photosensitizer (PS) that is highly phototoxic toa broad spectrum of bacteria (Cincotta et al., Photochem. Photobiol.,1987, 46:751-8). Recently, the present inventors have synthesized andcharacterized derivatives of the phenothiazine EtNBS that arefunctionalized for conjugation to substrates of hydrolytic enzymesproduced specifically by pathogens and not by the host (e.g., humans)(see FIG. 7). When the substrate is intact, the proximity of the PSmolecules to each other results in static quenching that is defined byreduced light absorbance and energy transfer capacities. Following theenzymatic cleavage of the substrate molecule, the PS molecules areseparated and therein released from quenching. When free, the PSexhibits increased absorbance and energy transfer capacity. The resultis a PS prodrug that is activated in and around the infectious drugresistant bacteria by a bacterial virulence enzyme and/or which iscapable of releasing a fluorescence signal when light-activated.

This example describes the use of a photosensitizer composition(“β-LEAPP”) comprising two EtNBS molecules conjugated to cephalosporin,a substrate molecule for beta-lactamase (see FIG. 8). The synthesisscheme depicted in FIG. 8 can be summarized as follows:7-amino-3-(4-aminophenylthio)methyl-3-cephem-4-carboxylic acidp-methoxybenzyl ester (1). ACLE hydrochloride (100 mg, 0.247 mmol) wassuspended in dichloromethane (DCM, 3 ml) at 0° C. Triethylamine (40 ul,0.287 mmol) was added in three portions over a 20 min period.4-methylmorpholine (NMM, 35 ul, 0.032 mmol) was added, followed with4-aminothiophenol (32 ul, 0.3 mmol). Reaction mixture was stirred at 0°C. for 2 h then purified using silica gel chromatography (1.5% methanolin DCM as eluent) to afford 46 mg (40%) of 1 as white solid. EtNBS-ACLEConjugates with p-methoxybenzyl protection group (3 and 4). EtNBS—COOH(130 mg, 0.3 mmol) and HATU (114 mg, 0.3 mmol) were stirred in DMF (500ul) for 30 min and 1 (46 mg, 0.1 mmol) was added to this reactionmixture. The reaction mixture was stirred at 4° C. for 72 h to allow forcomplete conversion of mono-substituted intermediate (2) todi-substituted product (3). The solvent was removed in vacuo and theresidue was redissolved in dichloromethane. The organic layer was washedwith brine and dried over sodium sulfate. After removing the solvent,the crude product was purified on silica preparative PLC (10% methanolin DCM as eluent) to afford 27 mg of di-EtNBS conjugate 3 (21%) and 26mg of mono-EtNBS conjugate 2 (30%). Compound 3 (6 mg, 0.0046 mmol) wasfurther dissolved in a mixture of trifluoroacetic acid, anisole and DCM(1.5 ml, 1:1:5) and stirred at 0° C. for 2 h. The solvent was removed invacuo and the residue was purified by RP-HPLC to yield 4 (5 mg, 90%yield).

Beta-lactamase is a hydrolytic enzyme expressed by many antibioticresistant bacteria and not by humans. Initial studies by the inventorshave demonstrated that the beta-lactamase enzyme activatedphotosensitizer prodrug (β-LEAPP) exhibits increased in vitroantibacterial PDT effect against MRSA when compared to a betalactamasenon-producing S. aureus (unpublished data). These findings promiseadvantage for the use of the prodrug in the PDT of antibiotic resistantbacterial infections, e.g., for use in the treatment ofMethicillin-Resistant Staphylococcus aureus (MRSA) and other drugresistant bacterial infections.

An advantageous feature of β-LEAPP is that the hydrolytic cleavage thatresults in its activation also results in the increase of itsfluorescence emission. In experimentation towards evaluating the abilityof various beta-lactamase producing strains of bacteria to activate,(cleave), β-LEAPP, it was discovered that the resultant increase influorescence emission was rapidly detectable and provided quantitativedata. This finding indicated that β-LEAPP is ideal for the detection andquantitation of beta-lactamase activity, amoung other uses.

Detection and Quantitation of Beta-Lactamase Activity

The ability of commercially available Penicillinase to hydrolyze theβ-LEAPP substrate was characterized in FIG. 9. The plots of fluorescenceunits as a function of time demonstrate the dependence of β-LEAPPfluorescence on Penicillinase concentration. Within the first 20 minutesof incubation the slopes of all of the plots are significantly differentfrom one another (see FIG. 9). From this data, a standard curve wasgenerated (see FIG. 10) through plotting the reciprocal of theinstantaneous velocity of the increase in β-LEAPP fluorescence (1/Vi) asa function of the reciprocal of the concentration of Penicillinase inunits per milliliter (1/U*ml-1). The instantaneous velocity (Vi) is therate of change in fluorescence within the region of the curve where theslope is linear. The linear region of the curve depends on theconcentration of enzyme. For the generation of a standard curve forβ-LEAPP fluorescence as a function of Penicillinase concentration thefirst 20 readings, taken over the first 20 minutes proved sufficientyielding an R² value of 0.9969 for the linear fit (see FIG. 10). Theequation that corresponds to the linear trend line of the doublereciprocal plot can be used to determine the beta-lactamase activitypresent in experimental samples where y equals the reciprocal of the Viand x equals the reciprocal of U (see FIG. 10 and Table 1 below).

The ability of various strains of bacteria, beta-lactamase producers,betalactamase non-producers, Gram positive, and Gram negative, tohydrolyze β-LEAPP was determined under conditions identical to thoseused for the assay of the hydrolytic cleavage by Penicillinase (seeFIGS. 11A and 11B). Beta-lactamase activity was detectable in culturesof both Gram positive and Gram negative bacteria. Only those bacterialstrains that produced beta-lactamase resulted in an increase influorescence emission when in incubation with β-LEAPP (see FIGS. 11A and11B).

The difference in the level of fluorescence emission produced bybeta-lactamase producing and non-producing strains of bacteria is clear(as shown in FIGS. 11A and 11B). All of the beta-lactamase producingstrains of MRSA and the ESBL producing strain of Escherichia coli showeda dramatic increase in fluorescence emission when compared to thatproduced by the beta-lactamase non-producing strains. The increase influorescence emission due to the incubation of β-LEAPP with thebeta-lactamase non-producing bacteria was identical to that produced byreaction buffer alone indicating that the marginal increase was due theminor degredation of β-LEAPP when under the reaction conditions.

TABLE 1 The values of the instanteous velocity (Vi) of β-LEAPPfluorescence emission produced by various beta-lactamase producingbacteria were used to calculate the amount of beta-lactamase activitypresent in the bacterial cultures. Slope (m) or 1/Units Units Strain(Vi) 1/Vi activity * ml⁻¹ Activity/ml MRSA 8179 0.635756 1.572930.811415 1.232415 MRSA 8150 0.4423 2.260909 1.266883 0.789339 MRSA 93070.466859 2.141973 1.188143 0.841649 ATCC 0.338173 2.957069 1.7277680.578782 BAA196

Discussion

There are over 500 different beta-lactamases known and over 200 of themare extended-spectrum-beta-lactamases (ESBLs) (Paterson et al., Clin.Microbio. Rev. 2005, 18:657-686). Commercially available substrates forthe detection of bacterial beta-lactamase produce a colorimetric changeupon hydrolysis where the color of the substrate changes hue orintensity. The substrates nitrocefin and centa, for example, bothexhibit a colorimetric change when hydrolyzed by beta-lactamase. Theformer, nitrocefin, is distributed in the form of impregnated paperdiscs. The application of a bacterial colony to a disc results in thedevelopment of a pink color. These prior known methods providequalitative (yes/no) detection, but do not provide any informationregarding the relative amount of enzymatic activity

or any insight into the type of beta-lactamase activity. The amount ofbeta-lactamase activity and the substrate specificity of that activityare important considerations in determining the appropriate antibiotictherapy for patients suffering from drug resistant bacterial infections.The nitrocephin disk test alone does not provide sufficient informationto determine the appropriate course of therapy in a clinical setting.Centa is distributed in the form of a powdered salt and also providesqualitative information, at a more reasonable cost, but requires theextended incubation of broth cultures for the development of color.

The enzymatic hydrolysis of β-LEAPP, in accordance with an embodiment ofthe present invention, results in an increase in fluorescence emission,thus providing a more sensitively detectable change than that ofcolorimetric substrates. This feature allows for the rapidquantification of enzyme activity and also

has the potential for use in the identification of the substratespecificity of various betalactamase enzymes. The use of β-LEAPP forthese purposes offers significant advantages not only for the researcherof bacterial beta-lactamase, but also for the clinical characterizationbeta-lactamase activity in that it provides more useful information. Theresearcher can determine the amount beta-lactamase activity and thesubstrate specificity of that activity simultaneously whereas thecurrent commercially available substrates provide only a qualitativeanswer. The assay time to determine the amount of enzyme activitypossessed by a particular strain of beta-lactamase producing bacteriacan be within about 20 minutes or even less. Through the inclusion ofvarious beta-lactams in the assay buffer one creates a competitionbetween β-LEAPP and the beta-lactam for binding the active site of theenzyme. Through comparison of the fluorescence emission over timebetween the competitive and non-competitive reactions the researcher candetermine whether or not a particular infectious bacteria is capable ofhydrolyzing the competitor substrate. The use of β-LEAPP in a clinicaldiagnostic role could provide the research with the valuable informationof whether or not the infectious bacteria has the capacity to hydrolyzeextended spectrum cephalosporins in a relatively short amount of time.This information is critical for the clinician to determine theappropriate course of antibiotic therapy and could result in improvedpatient outcomes through reducing the amount of time required for suchevaluations.

Example 4 Synthesis of β-LEAPP

This example describes the synthesis of a photosensitizer composition(“β-LEAPP”) comprising two EtNBS molecules conjugated to cephalosporin,a substrate molecule for beta-lactamase (see FIG. 8).

Rationale

The photosensitivity of phenothiazine is advantageously quenched untilreleased from the cephalosporin linked quencher in the presence ofβ-lactamase. Two quenching mechanisms are widely acknowledged. One isstatic (ground-state) quenching, achieved by homo- or hetero-fluorophoredimerization. The other is dynamic (excited-state) quenching, achievableby Forster resonance energy transfer (FRET). In this example, thephotosensitizer composition, β-LEAPP, was synthesized using a staticquenching mechanism. However, the same design can also be easily adaptedto FRET quenching.

Homo-dimerization (PS—PS pair) was advantagous in this example overhetero-dimeriation (PS-quencher pair) for at least the following tworeasons: 1) it is readily synthesized; 2) cleavage of each prodrug(β-LEAPP) will generate two PSs therefore in theory can achieve twicethe phototoxicity and/or fluorescence signaling.

The photosensitizing component in β-LEAPP (also referred to herein as“prodrug 2”) is5-(4′-carboxybutylamino)-9-diethylaminobenzo[a]phenothiazine(EtNBS—COOH), which was developed by the inventors and was shown to haveexcellent photosensitizing efficacy. The terminal carboxyl group withthe flexible alkyl chain provides an ideal site for conjugation.7-amino-3-chloromethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester(ACLE, commercial available) is used as cephalosporin scaffold. ACLE isa common starting material to synthesize cephalosporin derivatives. Itintegrates an amino group at 7-position. By replacing chlorine at3′-position with 4-amino-thiophenol provides an additional amino grouptherefore each ACLE molecule can accommodate two EtNBS-COOH molecules toform a static quenching pair.

Instrumentation

¹H and ¹³C-NMR were recorded on Varian 400 MHz instrument, using 15-20mg of material in CDCl3 or DMSO-d6 as solvents. ESI mass spectra wererecorded with a Bruker Daltonics Esquire 3000 plus spectrometer.UV-visible absorption spectra were recorded using a Hewlett Packard 8453spectrophotometer equipped with a diode array detector system.Fluorescence spectra were obtained in aqueous/organic solutions ofproducts using Jobin Yvon Horiba FluoroMax-3 fluorometer. HPLC analysiswere performed using Shimadzu VP series of SCL 10A controller, SPD-M10Adiode array detector, LC-LOAD pumps, DGU-14A degasser and C18 reversephase column controlled by Class VP software.

Synthesis and Characterization of β-LEAPP

Commercially available ACLE is a hydrochloride salt. Mild base as usedto treat the ACLE, resulting in the freeing of the amino group at7-position which is readily undergoing acylation. The chlorine at3′-position of ACLE was displaced by 4-aminothiophenol. As an excellentleaving group, the thiophenol residue facilitated the fragmentationafter β-lactamase hydrolysis, and the amino group on thiophenolintroduced an additional coupling site. EtNBS—COOH was coupled to both7- and 3′-amino in the presence ofO-(7-Azabenzotriazole-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HATU) as coupling reagent. A subsequent treatmentwith TFA in the presence of anisole gave unprotected prodrug 2 inexcellent yield. Purification of prodrug was carried on RP-HPLC.Characterization of prodrug 2 used NMR spectroscopy and massspectrometry.

Quenching Phenomena

The concentration of constituents in prodrug conjugates was routinelyscanned by absorption spectroscopy (between 200 nm and 800 nm) on diluteequimolar solutions of starting materials (EtNBS—COOH and ACLE) andsynthesized prodrug. Fluorescence quenching phenomena in prodrug withrespect to EtNBS—COOH was demonstrated using fluorescence emissionexperiments. Equimolar solutions of EtNBS-COOH and prodrug were comparedat 655 nm excitation with emissions scanned in the range of 655 nm to800 nm.

Lactamase Controlled Photosensitizer Release

Two commercial enzymes, a β-lactamase from Enterobacter cloacae and apenicillinase from Bacillus cereus were used to demonstrate release anddequenching. For this study, solutions of prodrugs were treated withrequired units of both enzymes. Time dependent fluorescence emission,indicating release of photosensitizer from conjugate, will be recordedand compared to free photosensitizer. Released photosensitizer willfurther be analyzed for its photosensitivity using Invitrogen's reactiveoxygen species detection kit (Posner et al., Biochem. Biophys. Res.Commun., 1984, 123:869-873; Thompson et al., Methods Enzymol., 1986,133:569-584; Li et al., J. Biol. Chem., 1998, 283:2015-2023) or usingdihydroethidium as an oxidizing probe (Dolgachev et al., Biochem.Biophys. Res. Commun., 2005, 332:411-417).

Results Anticipated

It is anticipate that β-LEAPP will achieve satisfied yield at more than95% purity. Due to very short distance between two photosensitizers,high quenching efficiency is anticipated. β-LEAPP is also anticipated toshow efficient recovery in excited state properties (such asfluorescence, singlet oxygen yield, cytotoxic effect) upon enzymaticcleavage/photosensitizer release.

Example 4 (Prophetic) Use of β-LEAPP to Determine Substrate Specificityof a Beta-Lactamase Activity of a Sample

The following example describes how to determine the specificity of agiven beta-lactamase enzyme.

Methods

Any suitable multiwell optically transparent plate i.e. 96 well cultureplate can be used according to this example to carry out the reactionsto determine enzyme substrate specificity. A useful configuration isdescribed below:

Set-up: “Criss-Cross Serial Dilution”

Serial concentrations of β-LEAPP are made from left to right across theplate or from top to bottom across the plate. Serial concentrations ofCompetitor Substrates are deposited from left to right across the plateor from top to bottom across the plate: These preferably include allbeta-lactam antibiotics. Serial concentrations of Beta-lactamaseinhibitors from left to right across the plate or from top to bottomacross the plate: These include clavulanic acid, sulbactam, andtazobactam

Add Sample and Monitor Fluorescence:

The rate of fluorescence emission change as a function of time for eachexperimental condition would be compared and the specificity of theenzyme would be determined.

Example 5 Use of β-LEAPP to Determine D-Lactamase Functionality in aCompetitive Substrate Inhibition Assay

Due to overlapping and similar characteristics of many beta-lactamases,it has become important and critical to the development of appropriateantibiotic regimens to have a means of distinguishing between differentbeta-lactamases associated with infectious bacterial agents, e.g.,multi-drug resistant S. aureus. It is well established thatbeta-lactamases can be characterized by their interactions withinhibitors and substrates (see Molecular Bacteriology: protocols andclinical applications, Ed. Woodford et al., Humana Press 1998, Chapter26: Biochemical and Enzyme Kinetic Applications for the Characterizationof β-lactamases, David J. Payne and Tony H. Farmer, pp. 513-537,incorporated herein by reference). For example, beta-lactamases can becharacterized based on their rates of hydrolysis of differentbeta-lactam substrates (e.g., penicillin, ampicillin, etc.) at fixedconcentrations or based on determining the I₅₀ (the concentration of aninhibitor that inhibits the hydrolytic activity of a beta-lactamase by50% compared with a control).

Other known methods relate to determining the susceptibility (e.g.,Minimum Inhibitory Concentrations—MIC) of bacteria to individualbeta-lactams, followed by determining their susceptibility tocombinations of beta-lactams (as recommended by the Clinical LaboratoryInstitute). Such an approach involves and, in fact, requires bacterialgrowth, and, thus, requires at least about 20 hours to achieve reliableresults. This type of approach is further described in Farber et al.,2008, “Extended-spectrum Beta-lactamase detection with different panelsfor automated susceptibility testing and with a chromogenic medium,” JClin Microbiol 46:3721-7 and Spanu et al., 2006, “Evaluation of the newVITEK 2 extended-spectrum beta-lactamase (ESBL) test for rapid detectionof ESBL production in Enterobacteriaceae isolates,” J Clin Microbiol,44:3257-62, each of which are incorporated herein by reference.

Due to the fact that many of the standard techniques for characterizingbeta-lactamases are typically slow and/or require bacterial growth,methods that are sensitive, rapid and easy to use would be desirable.

This Example describes a new methodology for use in characterizingbeta-lactamases by using a competitive substrate inhibition assay. It isbelieved that no competitive substrate inhibition assays for determiningbeta-lactamase functionality are currently in use by clinicalmicrobiology laboratories.

This new approach is based on exploiting the sensitivity and mechanismof the β-LEAP compound of the invention. More in particular, thisapproach is based on measuring the level of induced-fluorescence causedby the cleavage/hydrolysis and consequent activation of the β-LEAPcompound by beta-lactamase enzymes under evaluation while also in thepresence of, or “in competition” with, different types and amounts ofbeta-lactamase enzyme substrates. The degree of inhibition of β-LEAPhydrolysis/fluorescence due to competition with the beta-lactamcompetitor substrates is determined, which provides the basis fordetermining beta-lactamase functionality.

Methods for making the 13-LEAP of the invention and itsquenching/activation properties are described elsewhere in thisapplication (e.g., see Example 3) and by the inventors in Zheng et al.,2009, “Exploiting a bacterial drug-resistance mechanism: alight-activated construct for the destruction of MRSA,” Angew Chem IntEd Engl, 48:2148-51, which is incorporated herein be reference in itsentirety.)

This approach involved combining in a 384 multi-well assay plate formatpurified B. cereus beta-lactamase or bacterial suspensions with the13-LEAP of the invention and a competitive beta-lactam substrate,including amoxicillin, clavulanic acid, ampicillin, penicillin G,carbenicillin, cefazolin, cefatoxime or ceftazidime. For both purifiedB. cereus beta-lactamase enzyme and whole bacteria suspensions, thecephalosporins (ceftazidime, cefatoxime, and cephalothin) were moreeffective inhibitors of β-LEAP hydrolysis/fluorescence than were thepenicillins (amoxicillin, ampicilin, penicillin G, and carbenicillin)(FIG. 13a , which shows the inhibition constants (Ki) of a panel ofbeta-lactams for the competitive substrate inhibition of β-LEAPhydrolysis by B. cereus beta-lactamase; and FIG. 13b , which shows theinhibition constants (Ki) of a panel of beta-lactams for the competitivesubstrate inhibition of β-LEAP hydrolysis by bacterial suspensions).These results correlate with those obtained using a conventional MIC(Minimum Inhibitory Concentration) assay (see FIG. 13d , which shows theMICs of a panel of beta-lactams for B. cereus) and provided more usefulinformation. Importantly, the inhibitory effects of ampicillin andamoxicillin on β-LEAP hydrolysis were detected using a the β-LEAPcompetitive inhibition assay; however, neither ampicillin noramoxicillin resulted in any in vitro growth inhibition (FIG. 13d , noinhibition by amoxicillin, clavulanic acid or ampillin, data not shown).

These results indicate that β-LEAP may be used to identify thefunctionality of β-lactamase enzymes even where assays that rely onbacterial growth cannot. Accordingly, the competitive inhibition assaybased on inhibition of β-LEAP hydrolysis (i.e., decreased fluorescence)is a sensitive and rapid manner relative to previously known methods,e.g., MIC methods, by which to characterize the functionality of a givenbeta-lactamase enzyme from either a purified source or from a samplebacterial suspension.

What is claimed is:
 1. A photosensitizer composition, wherein thecomposition comprises at least one benzophenothiazinium chloride (EtNBS)photosensitizer conjugated to a cephalosporin linker or fragmentthereof.
 2. The photosensitizer composition of claim 1, wherein thecomposition comprises two benzophenothiazinium chloride (EtNBS)photosensitizers conjugated to a cephalosporin linker or fragmentthereof.
 3. The photosensitizer composition of claim 2, wherein at leastone photosensitizer is bound at the 3′ position of a cephalosporin. 4.The photosensitizer composition of claim 1, wherein the compositionfurther comprises a targeting moiety.
 5. The composition of claim 4,wherein the targeting moiety targets the composition to a pathogen or ahost cell infected with a pathogen.
 6. The composition of claim 5,wherein the infected host cell is a macrophage.
 7. The composition ofclaim 5, wherein the targeting moiety comprises a liposome.
 8. Thecomposition of claim 5, wherein the targeting moiety comprises apeptide.
 9. The composition of claim 8, wherein the peptide is a smallanti-microbial peptide or an active fragment or analog thereof.
 10. Thephotosensitizer composition of claim 2, further comprising one or morebinders effective to quench photoactivation of the benzophenothiaziniumchloride (EtNBS).
 11. The photosensitizer composition of claim 10,wherein the binder is a fluorophore.
 12. The photosensitizer compositionof claim 11, wherein the binder is a photosensitizer.
 13. Thephotosensitizer composition of claim 2, further comprising a backbonecoupled to the two benzophenothiazinium chloride (EtNBS) and one or morebinders effective to quench photoactivation, wherein the binders areconnected to the backbone through the linker.
 14. The photosensitizercomposition of claim 2, further comprising a backbone coupled to the twobenzophenothiazinium chloride (EtNBS) and one or more binders effectiveto quench photoactivation, wherein the photosensitizers are connected tothe backbone through the linker.
 15. The photosensitizer composition ofclaim 13 or 14, wherein the backbone comprises a targeting moiety. 16.The photosensitizer composition of claim 13 or 14, wherein the backbonecomprises a polyamino acid.
 17. The photosensitizer composition of claim16, wherein the polyamino acid is polylysine.
 18. A photosensitizercomposition according to formula I:X-L-X′, wherein L is a cephalosporin linker or fragment thereof, X isbenzophenothiazinium chloride (EtNBS) and X′ is benzophenothiaziniumchloride (EtNBS), wherein the photosensitizers are in a quenched statewhen L is uncleaved.
 19. The photosensitizer composition of claim 18,wherein the composition further comprises a targeting moiety.
 20. Thecomposition of claim 19, wherein the targeting moiety targets thecomposition to a pathogen or a host cell infected with a pathogen. 21.The composition of claim 20, wherein the infected host cell is amacrophage.
 22. The composition of claim 19, wherein the targetingmoiety comprises a liposome.
 23. The composition of claim 19, whereinthe targeting moiety comprises a peptide.
 24. The composition of claim23, wherein the peptide is a small anti-microbial peptide or an activefragment or analog thereof.
 25. The photosensitizer composition of claim18, further comprising one or more binders effective to quenchphotoactivation.
 26. The photosensitizer composition of claim 25,wherein the binder is a fluorophore.
 27. The photosensitizer compositionof claim 25, wherein the binder is a photosensitizer.
 28. Thephotosensitizer composition of claim 18, further comprising a backbonecoupled to the plurality of photosensitizers and one or more binderseffective to quench photoactivation, wherein the binders are connectedto the backbone through the linker.
 29. The photosensitizer compositionof claim 18, further comprising a backbone coupled to the plurality ofphotosensitizers and one or more binders effective to quenchphotoactivation, wherein the photosensitizers are connected to thebackbone through the linker.
 30. The photosensitizer composition ofclaim 28 or 29, wherein the backbone comprises a targeting moiety. 31.The photosensitizer composition of claim 28 or 29, wherein the backbonecomprises a polyamino acid.
 32. The photosensitizer composition of claim31, wherein the polyamino acid is polylysine.
 33. A method for detectinga beta-lactamase activity in a sample, comprising the steps of:contacting the sample with a photosensitizer composition comprising twoor more photosensitizers that are conjugated to a linker, wherein thelinker comprises a cleavage site for a beta-lactamase and wherein thephotosensitizers are quenched when the cleavage site is intact butunquenched when the cleavage site is hydrolyzed; detecting cleavage ofsaid linker, wherein cleavage of said linker is indicative of abeta-lactamase activity in the sample.
 34. The method of claim 33,wherein detecting an unquenched photosensitizer comprises detecting asignal produced by the unquenched photosensitizer.
 35. The method ofclaim 34, wherein the signal is a fluorescence emission induced byilluminating the unquenched photosensitizer with an excitationwavelength.
 36. The method of claim 33, wherein the sample is abiological sample isolated from an infection in a subject.
 37. Themethod of claim 36, wherein the subject is a mammal.
 38. The method ofclaim 37, wherein the mammal is a human.
 39. The method of claim 36,wherein the infection is caused by an antibiotic-resistant pathogen. 40.The method of claim 39, wherein the antibiotic-resistant pathogen is aGram (−) bacterium or Gram (+) bacterium.
 41. The method of claim 39,wherein the antibiotic-resistant pathogen is selected from the groupconsisting of Staphylococcus, Enterococcus, Enterobacter, Escherichia,Haemophilus, Neisseria, Klebsiella, Pasteurella, Proteus, Pseudomonas,Streptophomonas, Burkholderia, Acinetobacter, Serratia, and Salmonellaspp.
 42. The method of claim 39, wherein the antibiotic-resistantpathogen is selected from the group consisting of Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, and Salmonellatyphimurium.
 43. The method of claim 39, wherein the photosensitizer isa porphryin selected from the group consisting of a porfimer sodium,hematoporphyrin IX, hematoporphyrin ester, dihematoporphyrin ester,synthetic diporphyrin, O-substituted tetraphenyl porphyrin, 3,1-mesotetrakis porphyrin, hydroporphyrin, benzoporphyrin derivative,benzoporphyrin monoacid derivative, monoacid ring derivative,tetracyanoethylene adduct of benzoporphyrin, dimethylacetylenedicarboxylate adduct of benzoporphyrin, δ-aminolevulinic acid,benzonaphthoporphyrazine, naturally occurring porphyrin, ALA-inducedprotoporphyrin IX, synthetic dichlorin, bacteriochlorintetra(hydroxyphenyl) porphyrin, purpurin, octaethylpurpurin derivative,etiopurpurin, tin-etio-purpurin, porphycene, chlorin, chlorin e₆,mono-1-aspartyl derivative of chlorin e₆, di-1-aspartyl derivative ofchlorin e₆, tin(IV) chlorin e₆, meta-tetrahydroxyphenylchlorin, chlorine₆ monoethylendiamine monamide, verdin, zinc methyl pyroverdin, copro IIverdin trimethyl ester, deuteroverdin methyl ester, pheophorbidederivative, pyropheophorbide, texaphyrin, lutetium (III) texaphyrin, andgadolinium(III) texaphyrin.
 44. The method of claim 39, wherein thephotosensitizer is a photoactive dye selected from the group consistingof a merocyanine, phthalocyanine, chloroaluminum phthalocyanine,sulfonated aluminum PC, ring-substituted cationic PC, sulfonated AlPc,disulfonated or tetrasulfonated derivative, sulfonated aluminumnaphthalocyanine, naphthalocyanine, tetracyanoethylene adduct, crystalviolet, azure β chloride, benzophenothiazinium, benzophenothiaziniumchloride (EtNBS), phenothiazine derivative, rose Bengal, toluidine bluederviatives, toluidine blue O (TBO), methylene blue (MB), new methyleneblue N (NMMB), new methylene blue BB, new methylene blue FR,1,9-dimethylmethylene blue chloride (DMMB), methylene blue derivatives,methylene green, methylene violet Bernthsen, methylene violet 3RAX, Nileblue, Nile blue derivatives, malachite green, Azure blue A, Azure blueB, Azure blue C, safranine O, neutral red,5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride,5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride,thiopyronine, and thionine.
 45. The method of claim 39, wherein thephotosensitizer composition comprises two benzophenothiazinium chloride(EtNBS) photosensitizers conjugated to a cephalosporin linker orfragment thereof.
 46. The method of claim 35, further comprisingquantitating the signal produced by the unquenched photosensitizer. 47.The method of claim 46, wherein the step of quantitating the signalcomprises measuring the amount of the signal with a detector.
 48. Amethod for determining the substrate specificity of a beta-lactamaseenzyme in a sample, comprising: contacting the sample with aphotosensitizer composition comprising two photosensitizers that areconjugated to a linker, wherein the linker comprises a cleavage site fora beta-lactamase enzyme and wherein the photosensitizers are quenchedwhen the cleavage site is intact but unquenched when the cleavage siteis cleaved; determining whether the linker is cleaved; wherein cleavageof the linker indicates the linker is a substrate of the beta-lactamaseenzyme.
 49. The method of claim 48, wherein the step of determiningwhether the linker is cleaved further comprises detecting a first signalproduced by the unquenched photosensitizers.
 50. The method of claim 49,wherein the first signal comprises a fluorescence emission induced byilluminating the unquenched photosenstitizers with an excitationwavelength.
 51. The method of claim 49, further comprising carrying outa second reaction comprising: contacting the sample with thephotosensitizer composition and a beta-lactam antibiotic; detecting asecond signal produced by the unquenched photosensitizer of said secondreaction; and comparing the first and second signals, wherein thebeta-lactam antibiotic is identified as a substrate to thebeta-lactamase enzyme when the first signal is larger than the secondsignal.
 52. The method of claim 51, administering to a subject in need abeta-lactam antibiotic that is not identified as a substrate of thebeta-lactamase enzyme.
 53. The method of claim 48, wherein the sample isa biological sample isolated from an infection of the subject in needthereof.
 54. The method of claim 53, wherein the subject is a mammal.55. The method of claim 54, wherein the mammal is a human.
 56. Themethod of claim 53, wherein the infection is caused by anantibiotic-resistant pathogen.
 57. The method of claim 56, wherein theantibiotic-resistant pathogen is a Gram (−) bacterium or Gram (+)bacterium.
 58. The method of claim 56, wherein the antibiotic-resistantpathogen is selected from the group consisting of Staphylococcus,Enterococcus, Enterobacter, Escherichia, Haemophilus, Neisseria,Klebsiella, Pasteurella, Proteus, Pseudomonas, Streptophomonas,Burkholderia, Acinetobacter, Serratia, and Salmonella spp.
 59. Themethod of claim 56, wherein the antibiotic-resistant pathogen isselected from the group consisting of Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, and Salmonellatyphimurium.
 60. A method of typing a beta-lactamase enzyme in a sample,comprising: performing a competitive reaction comprising the steps of(a) contacting the sample with a photosensitizer composition comprisingtwo photosensitizers that are conjugated to a linker, wherein the linkercomprise a cleavage site for a beta-lactamase and wherein thephotosensitizers are in a quenched state; (b) cleaving the linker todequench the photosensitizers; (c) light-activating the composition toproduce a fluorescence signal; and (d) quantifying the fluorescencesignal with a detector to obtain a result, said competitive reactionbeing performed in the presence of a competing beta-lactam antibiotic;comparing the result of the competitive reaction to a standard to typethe beta-lactamase.
 61. The method of claim 60, wherein the standard isdetermined by performing a non-competitive reaction comprising the stepsof (a) contacting the sample with a photosensitizer compositioncomprising two photosensitizers that are conjugated to a linker, whereinthe linker comprise a cleavage site for a beta-lactamase and wherein thephotosensitizers are in a quenched state; (b) cleaving the linker todequench the photosensitizers; (c) light-activating the composition toproduce a fluorescence signal; and (d) quantifying the fluorescencesignal with a detector to obtain a standard.
 62. The method of claim 60,wherein the sample is a biological sample isolated from an infection ofthe subject in need thereof.
 63. The method of claim 62, wherein thesubject is a mammal.
 64. The method of claim 63, wherein the mammal is ahuman.
 65. The method of claim 62, wherein the infection is caused by anantibiotic-resistant pathogen.
 66. The method of claim 65, wherein theantibiotic-resistant pathogen is a Gram (−) bacterium or Gram (+)bacterium.
 67. The method of claim 65, wherein the antibiotic-resistantpathogen is selected from the group consisting of Staphylococcus,Enterococcus, Enterobacter, Escherichia, Haemophilus, Neisseria,Klebsiella, Pasteurella, Proteus, Pseudomonas, Streptophomonas,Burkholderia, Acinetobacter, Serratia, and Salmonella spp.
 68. Themethod of claim 65, wherein the antibiotic-resistant pathogen isselected from the group consisting of Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, and Salmonellatyphimurium.
 69. A kit for detecting a beta-lactamase activity in asample comprising a photosensitizer composition of claim 1 andinstructions for using the photosensitizer composition to detect abeta-lactamase activity in a sample.
 70. The kit of claim 69, whereinthe sample is a biological sample isolated from an infection of asubject.
 71. The kit of claim 70, wherein the subject is a mammal. 72.The kit of claim 71, wherein the mammal is a human.
 73. The kit of claim70, wherein the infection is caused by an antibiotic-resistant pathogen.74. The kit of claim 73, wherein the antibiotic-resistant pathogen is aGram (−) bacterium or Gram (+) bacterium.
 75. The kit of claim 73,wherein the antibiotic-resistant pathogen is selected from the groupconsisting of Staphylococcus, Enterococcus, Enterobacter, Escherichia,Haemophilus, Neisseria, Klebsiella, Pasteurella, Proteus, Pseudomonas,Streptophomonas, Burkholderia, Acinetobacter, Serratia, and Salmonellaspp.
 76. The kit of claim 75, wherein the antibiotic-resistant pathogenis selected from the group consisting of Staphylococcus aureus,Staphylococcus epidermis, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, Haemophilus influenzae, Neisseria gonorrhea,Klebsiella pneumoniae, Pasteurella multocida, Proteus mirabilis,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderiacepacia, Acinetobacter baumannii, Enterobacter aerogines, Enterobactercloacae, Serratia marcescens, Salmonella enterica, and Salmonellatyphimurium.
 77. A kit for determining the substrate specificity of abeta-lactamase activity in a sample comprising the photosensitizercomposition of claim 1 and instructions for using the photosensitizercomposition to type a beta-lactamase activity in a sample.
 78. The kitof claim 77, wherein the sample is a biological sample isolated from aninfection of a subject.
 79. The kit of claim 78, wherein the subject isa mammal.
 80. The kit of claim 79, wherein the mammal is a human. 81.The kit of claim 77, wherein the infection is caused by anantibiotic-resistant pathogen.
 82. The kit of claim 81, wherein theantibiotic-resistant pathogen is a Gram (−) bacterium or Gram (+)bacterium.
 83. The kit of claim 82, wherein the antibiotic-resistantpathogen is selected from the group consisting of Staphylococcus,Enterococcus, Enterobacter, Escherichia, Haemophilus, Neisseria,Klebsiella, Pasteurella, Proteus, Pseudomonas, Streptophomonas,Burkholderia, Acinetobacter, Serratia, and Salmonella spp.
 84. The kitof claim 82, wherein the antibiotic-resistant pathogen is selected fromthe group consisting of Staphylococcus aureus, Staphylococcus epidermis,Enterococcus faecalis, Enterococcus faecium, Escherichia coli,Haemophilus influenzae, Neisseria gonorrhea, Klebsiella pneumoniae,Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa,Stenotrophomonas maltophilia, Burkholderia cepacia, Acinetobacterbaumannii, Enterobacter aerogines, Enterobacter cloacae, Serratiamarcescens, Salmonella enterica, and Salmonella typhimurium.
 85. Amethod of treating a bacterial infection in a subject in need,comprising administering a therapeutically effective amount of anantibiotic, wherein the antibiotic does not have the same structure as acleavable linker detected by the methods of claim 33, 48, or
 60. 86. Thephotosenstizer composition of claim 18, wherein the linker L apenicillin selected from the group consisting of: benzthine penicillin,benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V),procaine penicillin, oxacillin, methicillin, dicloxacillin,flucloxacillin, temocillin, amoxicillin, ampicillin, co-amoxiclav,carboxypenicillins, ureidopenicillins, azlocillin, carbenicillin,ticarcillin, mezlocillin, piperacillin, and any fragment or derivativeof the above.
 87. The photosenstizer composition of claim 18, whereinthe linker L is a cephalosporin selected from the group consisting of:Cefacetrile (cephacetrile), Cefadroxil (cefadroxyl; Duricef®), Cefalexin(cephalexin; Keflex®), Cephaloglycin, Cefalonium (cephalonium),Cefaloridine (cephaloradine), Cefalotin (cephalothin; Keflin®),Cefapirin (cephapirin; Cefadryl®), Cefatrizine, Cefazaflur, Cefazedone,Cefazolin (cephazolin; Ancef®, Kefzol®), Cefradine (cephradine;Velosef®), Cefroxadine, Ceftezole, Cefaclor (e.g., Ceclor®, Distaclor®,Keflor®, Raniclor®), Cefonicid (e.g, Monocid®), Cefprozil (e.g.,cefproxil; Cefzil®), Cefuroxime (e.g., Zinnat®, Zinacef®, Ceftin®,Biofuroksym®), Cefuzonam, Cefmetazole, Cefotetan, Cefoxitin,Carbacephems (e.g., loracarbef (Lorabid′)), Cephamycins (e.g.,cefbuperazone, cefmetazole (Zefazone®), cefminox, cefotetan (Cefotan®),cefoxitin (Mefoxin®)), cefotetan or cefoxitin, Cefcapene, Cefdaloxime,Cefdinir (Omnicef®), Cefditoren; Cefetamet, Cefixime (Suprax®),Cefmenoxime, Cefodizime, Cefotaxime (Claforan®), Cefpimizole,Cefpodoxime (Vantin®, PECEF), Cefteram, Ceftibuten (Cedax), Ceftiofur,Cefliolene, Ceftizoxime (Cefizax®), Ceftriaxone (Rocephin®),Cefoperazone (Cefobid), Ceftazidime (Forturn®, Fortaz®), or Oxacephems(e.g. latamoxef), cefepime (Maxipime®), cefclidine, cefluprenam,cefoselis, cefozopran, cefpirome, cefquinome, cefpirome, and a fragmentof derivative of any of the above.
 88. The photosensitizer compositionof claim 1, wherein the EtNBS photosensitizer is quenched when thelinker is uncleaved and unquenched when the linker is cleaved.
 89. Themethod of claim 60, wherein the competing beta-lactam antibiotic is acephalosporin.
 90. The method of claim 60, wherein the competingbeta-lactam antibiotic is a penicillin.